American Journal of Botany 94(9): 1554–1569. 2007.

PHYLOGENY OF THE TRIBE AVENEAE (,) INFERRED FROM PLASTID TRNT-F AND NUCLEAR ITS SEQUENCES1

ALEJANDRO QUINTANAR,2,4 SANTIAGO CASTROVIEJO,2 AND PILAR CATALA´ N3

2Real Jardı´n Bota´nico de Madrid, CSIC, Madrid, ; and 3Escuela Polite´cnica Superior de Huesca, Universidad de Zaragoza, Zaragoza, Spain

New insights into evolutionary trends in the economically important oat tribe (Aveneae) are presented. Plastid trnT-F and nuclear ribosomal ITS sequences were used to reconstruct the phylogeny of the Aveneae––Seslerieae complex (Pooideae, Poaceae) through Bayesian- and maximum parsimony-based analyses, separately and in combination. The plastid data identified a strongly supported core Aveneae lineage that separated from other former Aveneae and Poeae groups. Koeleriinae, Aveninae, and Agrostidinae emerged as the main groups of this core Aveneae, which also included other minor subgroups with uncertain relationships and a few former Poeae members. Several former Aveneae representatives were also placed in independent sublineages in Poeae. Seslerieae resolved as close allies of Poeae or Aveneae in the plastid and nuclear topologies, respectively. Because of the intermingling of some Aveneae and Seslerieae lineages in Poeae and vice versa, we propose to expand Poeae to include all the aforementioned lineages. This best reflects our current understanding of the phylogeny of these important temperate grasses and sheds light on their evolutionary history.

Key words: Aveneae; grass systematics and evolution; molecular phylogenetics; plastid and nuclear DNA sequence data; Poeae; Pooideae; Seslerieae.

The oat tribe, Aveneae Dumort. (including Agrostideae in temperate grassland formations in natural, less-disturbed Dumort.), is the second largest tribe in subfamily Pooideae areas (Clayton and Renvoize, 1986; Ro¨ser, 1997). Benth. and is one of the main groups of the grass family Aveneae classification and its taxonomical borders with its [Poaceae (R. Br.) Barnhart]. It includes the economically sister tribe Poeae R. Br. have varied historically depending on important oats, one of the most ancient food supplies for an author’s interpretations of the tribe’s morphologic hetero- humankind, and many of the most abundant grasses of geneity; consequently, the adscription of many of its genera has temperate ecosystems. It comprises about 57 genera and been problematical (Table 1). In modern classifications, 1050 (Clayton and Renvoize, 1986) that inhabit Aveneae have been separated from Poeae (and partly from temperate-to-arctic regions throughout the world (Stebbins, Seslerieae Koch) based on the floral traits cited (Tzvelev, 1976; 1956; Stebbins and Crampton, 1961; Clayton, 1975, 1981; MacFarlane and Watson, 1982; Clayton and Renvoize, 1986; MacFarlane and Watson, 1980, 1982; Clayton and Renvoize, Watson and Dallwitz, 1992). Tzvelev (1989), however, did not 1986; MacFarlane, 1987; Watson and Dallwitz, 1992). recognize Aveneae but transferred their members to the large Traditionally, the Aveneae have been characterized by tribe Poeae, although Phleeae Dumort. (including Phalarideae morphologic traits related to their archtypical spikelet form of Kunth) was separated from Poeae. An increasing number of long glumes (relative to spikelet length) and a tendency toward phylogenetic studies in recent decades have helped to clarify a reduced number of flowers per spikelet, commonly 1, 2, or 2– evolutionary relationships within the subfamily Pooideae 3 per spikelet. Other, apparently derived, features of Aveneae (Soreng et al., 1990; Davis and Soreng, 1993; Nadot et al., include a soft endosperm with lipid energy reservoirs, which 1994; Hsiao et al., 1995; Catala´n et al., 1997; GPWG, 2001). However, the details of the phylogeny of Aveneae have presumably has adaptive value. Most of these features have remained largely unexplored. Most phylogenetic surveys been interpreted as resulting from evolutionary trends that have related to the avenoids have focused on particular genera, like yielded highly specialized taxa (Clayton and Renvoize, 1986; (Grebenstein et al., 1998), (Rodionov et Ro¨ser, 1997). al., 2005), (S. Nisa et al., unpublished data), Another remarkable feature of the tribe is the inclusion of a and (Chiapella, 2007). large radiation of annual genera, mostly in the pan-Mediter- The first phylogenetic study with a large sampling of ranean region, adapted to arid conditions and disturbance. Aveneae taxa was by Soreng and Davis (2000), who also These annual species usually colonize ephemeral, often- explored the relationships of its sister tribe Poeae. Their disturbed habitats, whereas most of the perennial taxa grow combined analysis of plastid restriction site data and structural data resulted in a consensus topology where the sister 1 Manuscript received 26 June 2006; revision accepted 11 July 2007. divergence of the main Aveneae and Poeae lineages was The authors thank J. Mu¨ller, R. Soreng, and C. Stace for valuable blurred by several admixtures of misplaced genera of the comments and revision of an earlier version of the manuscript; L. A. Inda, opposite tribe. Genera traditionally classified in Poeae, such as E. Pe´rez, M. Pimentel, and J. G. Segarra for their support and assistance in , Chascolytrum Desv., Poidium Nees, and the laboratory; and J. Mu¨ller, P. M. Peterson, M. Pimentel, M. Sequeira, R. J. Soreng, and the curators of the JACA, MA, and US herbaria for G. L. Church, were resolved as closely related to different providing fresh samples and herbarium materials for the analyses. This Aveneae lineages. Conversely, other genera formerly recog- work was subsidized by the Ministerio de Ciencia y Tecnologı´a of Spain nized as Aveneae, like , Alopecurus, Holcus, and (projects REN2003-02818/GLO and REN2002-04634-C05-05). , were nested within different clades of Poeae. Finally, 4 Author for correspondence (e-mail: [email protected]) Soreng and Davis placed Aveneae within Poeae and recog- 1554 September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1555 nized a series of subtribes of Aveneae that were later expanded (except Duthieinae) were sampled and incorporated into the analysis. Our (Soreng et al., 2003). Past intertribal hybridization events were sampling includes representatives from subtribes Airinae Fr., Agrostidinae Fr., advocated as a plausible explanation for the present existence Alopecurinae Dumort., Anthoxanthinae A. Gray, Aveninae J. Presl, Beck- manniinae Nevski, Gaudiniinae Holub, Holcinae Dumort., Koeleriinae Asch. & of certain Avenae taxa with Poeae plastid genomes and vice Graebn., Miliinae Dumort., Phalaridinae Fr., Phleinae Dumort., and Ventena- versa, while most other cases were attributed to traditional tinae Holub (Appendix). The poorly studied subtribe Koeleriinae was more misclassifications (Soreng and Davis, 2000). The systematic exhaustively sampled in our study with a wide representation of the and evolutionary placement of the tribe Seslerieae with respect and Trisetum taxa, as well as representatives of their supposedly allied genera to Aveneae and Poeae has also been debated (Table 1). Avellinia, Dielsiochloa, , , , and Ven- Seslerieae includes several genera characterized by their tenata. Sampling of the sister tribe Poeae (32 taxa, 20 genera) included representatives of the main lineages of this group, i.e., the subtribes Loliinae strongly condensed , often subtended by glume- and its close allies Parapholiinae Caro, Cynosurinae Fr., and Dactylidinae like bracts. The few molecular studies on single representatives Stapf, and Poinae and its close ally Puccinelliinae Soreng & J. I. Davis, for of (Soreng and Davis, 2000; Catala´n et al., 2004; which new sequences were provided (Appendix), plus other lineages with an Gillespie et al., 2006) indicated that this was close to unexpectedly close relation to Aveneae (Briza) or an uncertain attribution and either Aveneae or Poeae, but the relationships were not relationships (Anthochloa, , Cinna, Scolochloa, etc.) (Table 1). satisfactorily resolved. Duthieinae Potzal, which was subsumed Representatives of Seslerieae (Tzvelev, 1976) (Table 1) also were included in our survey (three genera, four taxa; Appendix). Our systematic scheme follows by Clayton and Renvoize (1986) under Aveneae but is the tribal and subtribal circumscriptions proposed by Tzvelev (1976) and characterized by primitive traits such as havingthree lodicules Watson and Dallwitz (1992), and the generic ordering of Tutin et al. (1980). and three stigmas, was considered distantly related to Pooideae but rather close to Arundinoideae Burmeist. (Watson and DNA isolation, amplification, and sequencing—Leaf tissue from either Dallwitz, 1992) or Stipeae Dumort. (Soreng et al., 2003). fresh silica-gel-dried materials or herbarium vouchers was ground to powder in There is a current and increasing interest in the boundaries of liquid nitrogen. Total DNA was isolated from each sample following the Aveneae and the evolutionary relationships among Aveneae procedures of the DNeasy Mini Kit (Qiagen (Qiagen group, F. lineages and the closely allied Poeae and Seslerieae. Conse- Hoffmann—Spain Izasa S.A.)). The plastid trnT-F was amplified and sequenced separately for the trnT-L and trnL-F subregions using external quently, we initiated an extended phylogenetic survey of these primer pairs a-forward/b-reverse and c-forward/f-reverse, respectively, as groups using nuclear and plastid data. In the present study, we described by Taberlet et al. (1991). The primer pair combination fern-forward/ include 42 genera of Aveneae (56% of its generic diversity f-reverse (Torrecilla et al., 2003) also was used for trnL-F amplification. Fifty- sensu Watson and Dallwitz, 1992) and three genera of microliter PCR reactions were prepared with 5 lL103 buffer, 5 lL MgCl2 (3 Seslerieae. Of the main Poeae lineages, 20 genera are included mM), 2 lL dNTPs (10 mM), 1 lL forward primer (50 lM), 1 lL reverse primer (32% of the total). Our phylogenetic reconstructions are based (50 lM), 34.5 lL ddH2O, 0.5 lL Taq (1.5 u), and 1 lL DNA. Amplifications were performed as follows: one denaturing cycle of 60 s at 948C; 30 cycles on analyses of DNA sequences from both the plastid trnT-F including a 15-s denaturing at 948C, a 30-s annealing at 458C, and a 60-s region and the nuclear ribosomal ITS region (ITS1–5.8S- extension at 728C; followed by a termination step of 7 min at 728C. The ITS ITS2). Use of nuclear and organellar phylogenies is recognized region (ITS1–5.8S-ITS2) was amplified and sequenced using the external as a reasonably sound approach for understanding the history primer pair combinations KRC-forward/ITS4-reverse and ITS1-forward/ITS4- of groups that have presumably experienced reticulate reverse (Torrecilla and Catala´n, 2002). Fifty-microliter PCR reactions were evolution (Soltis and Kuzoff, 1995). The value of sequences prepared in a reaction mix that consisted of 5 lL103 buffer, 2 lL MgCl2 (3 of the plastid trnT-F region (trnT-L spacer and the useful trnL mM), 2.5 lL dNTPs (0.5 mM), 1.25 lL forward primer (50 lM), 1.25 lL 0 reverse primer (50 lM), 36.7 lL ddH2O, 0.3 lL Taq polymerase (1.5 u), and 1 intron–trnL 3 exon–trnL-F spacer) for resolving phylogenetic lL DNA. Amplifications were carried out under the following conditions: one relationships was shown in the separation of the main lineages denaturing cycle of 3 min at 948C; 35 cycles of a 1-min denaturing at 948C, a 1- of the large subtribes Loliinae Dumort. (Torrecilla et al., 2004; min annealing at 508C, and a 1-min extension at 728C; followed by a Catala´n et al., 2004) and Poinae Dumort. (Brysting et al., 2004; termination step of 7 min at 728C. All PCR products were purified with the Hunter et al., 2004) of Poeae. The ITS region has also been QIAquick PCR Purification Kit (Qiagen). When more than one PCR band was recovered, the suitable amplified band was separated on 1% agarose gels, shown to be informative for phylogenetic inference in several excised, and purified using the QIAquick Gel Extraction Kit (Qiagen). Clean Aveneae (Helictotrichon, Grebenstein et al., 1998; Avena, PCR products were sequenced with the BigDye Terminator v3.1 Cycle Rodionov et al., 2005; Deschampsia, Chiapella, 2007) and in Sequencing Kit (Applied Biosystems). Five-microliter sequencing reactions the subtribe Loliinae of Poeae (Charmet et al., 1997; Gaut et were prepared with 1.5 lL53 sequencing buffer, 1 lL sequencing mix, 1 lL al., 2000; Torrecilla and Catala´n, 2002; Torrecilla et al., 2004; primer, 1.5 lL ddH2O, and 5 lL purified DNA. PCR was performed with one Catala´n et al., 2004), mostly because of its biparental denaturing cycle of 1 min at 958C; 99 cycles of 10-s denaturing at 958C, a 5-s annealing at 508C, and a 4-s extension at 608C; then a termination step of 4 min inheritance, the coupled effect of concerted evolution (Baldwin at 608C. et al., 1995), and a moderate rate of mutation (Torrecilla and Catala´n, 2002) in temperate-climate grasses. By separate and Sequence alignments and phylogenetic analyses—The trnT-F, trnL-F, combined analysis of data from these two independent genomic and ITS sequences were aligned separately using the program ClustalX, sources, we aim to reconstruct a phylogeny that can be used as version1.83 (Thompson et al., 1997). Manual alignment was further performed a baseline to interpret the evolutionary trends of the highly on each data matrix with the aid of the program Se-Al v. 2.0a11 (Rambaud, relevant but inadequately explored oat tribe. 1996). The boundaries of the plastid trnL-F region and the nuclear ITS region were determined according to those established by Catala´n et al. (2004) for the subtribe Loliinae, and the boundaries of the plastid trnT-F region were determined according to those established by Mason-Gamer et al. (2002) for the MATERIALS AND METHODS tribe Triticeae Dumort. The concatenated trnT-F and trnL-F data sets were united into a single trnT-F plastid data matrix of taxa common to the two Plant material—Sampling was designed to be representative of the separate data matrices. A total of 75 new ITS sequences (GenBank accession taxonomic/phenotypic diversity in the Aveneae tribe and included 105 species numbers DQ336815–336834 and DQ539562–539616), 97 new trnT-L and subspecies of 42 genera from the main lineages thought to belong to this (DQ336855–336880, DQ367404–367407, and DQ631481–631547), and 73 tribe (Watson and Dallwitz, 1992). Generic representatives of all subtribes of new trnL-F sequences (DQ336835–336854 and DQ631428–631480) were Aveneae recognized by Tzvelev (1976) and by Clayton and Renvoize (1986) generated for this study. Another 67 ITS and 26 trnL-F sequences were 1556 AMERICAN JOURNAL OF BOTANY [Vol. 94

TABLE 1. Placement of the Aveneae and Seslerieae representatives included in our study in selected classification systems.

Ascherson and Graebner Clayton and Watson and (1898–1902) Maire et al. (1953) Prat (1960) Tzvelev (1976) Tutin et al. (1980) Renvoize (1986) Dallwitz (1992) Soreng et al. (2003)

Aveneae Aveneae Aveneae Aveneae Aveneae Aveneae Aveneae Poeae Airinae Airinae Aveninae Airinae Aira Aira Aira Aira Aira Aira Aira Corynephorus Corynephorus Corynephorus Corynephorus Corynephorus Corynephorus Corynephorus Deschampsia Deschampsia Deschampsia Deschampsia Deschampsia Deschampsia Holcinae Holcinae Holcus Holcus Holcus Holcus Holcus Holcus Holcus Airopsis Airopsis Airopsis Airopsis Airopsis Periballia Periballia Periballia Periballia Periballia Antinoria Antinoria Antinoria Aveninae Aveninae Aveninae Arrhenatherum Arrhenatherum Arrhenatherum Arrhenatherum Arrhenatherum Arrhenatherum Arrhenatherum Arrhenatherum Avena Avena Avena Avena Avena Avena Avena Avena Avenula/ Avenula/ Avenula/ Avenula/ Avenula/ Avenula/ Avenula/ Helictotrichon Helictotrichon Helictotrichon Helictotrichon Helictotrichon Helictotrichon Helictotrichon Helictotrichon Pseudarrhena- Pseudarrhena- Pseudarrhena- Pseudarrhena- therum therum therum therum Corynephorus Deschampsia Holcus Gaudiniinae Gaudinia Gaudinia Gaudinia Gaudinia Gaudinia Gaudinia Gaudinia Gaudinia Avellinia Koeleriinae Avellinia Avellinia Avellinia Koeleria Koeleria Koeleria Koeleria Koeleria Koeleria Koeleria Rostraria Rostraria Rostraria Rostraria Rostraria Rostraria Rostraria Trisetum Trisetum Trisetum Trisetum Trisetum Trisetum Trisetum Trisetum Dielsiochloa Dielsiochloa Dielsiochloa Graphephorum Graphephorum Graphephorum Ventenatinae Sphenopholis Sphenopholis Sphenopholis Ventenata Ventenata Ventenata Ventenata Ventenata Ventenata Ventenata Cinna Agrostideae Agrostideae Agrostideae Agrostidinae Agrostidinae Agrostidinae Alopecurinae Agrostidinae Agrostis Agrostis Agrostis Agrostis Agrostis Agrostis Agrostis Ammophila Ammophila Ammophila Ammophila Ammophila Ammophila Ammophila Apera Apera Apera Apera Apera Apera Apera Calamagrostis Calamagrostis Calamagrostis Calamagrostis Calamagrostis Calamagrostis Calamagrostis Gastridium Gastridium Gastridium Gastridium Gastridium Gastridium Gastridium Gastridium Lagurus Lagurus Lagurus Lagurus Lagurus Lagurus Lagurus Polypogon Polypogon Polypogon Polypogon Polypogon Polypogon Polypogon Zingeria Zingeria Zingeria Triplachne Triplachne Triplachne Triplachne Triplachne Chaetopogon Chaetopogon Chaetopogon Chaetopogon Chaetopogon Cinna Cinna Cinna Cinna Deyeuxia Deyeuxia Deyeuxia Miborinae Miborinae Ammochloa Mibora Mibora Mibora Mibora Mibora Mibora Deschampsia Phleeae Ventenata Phleinae Phleinae Alopecurinae Alopecurinae Alopecurus Alopecurus Alopecurus Alopecurus Alopecurus Alopecurus Alopecurus Alopecurus Phleinae Phleum Phleum Phleum Phleum Phleum Phleum Phleum Phleum Chlorideae Isolated Beckmanniinae Beckmannia Beckmannia Beckmannia Beckmannia Beckmannia Beckmannia Phalarideae Phalarideae Phalarideae Phalarideae (sub Panicoideae) Anthoxanthinae Phalaridinae Phalaridinae Anthoxanthum Anthoxanthum Anthoxanthum Anthoxanthum Anthoxanthum Anthoxanthum Anthoxanthum Anthoxanthum Hierochloe Hierochloe Hierochloe Hierochloe Hierochloe Hierochloe Hierochloe Phalaridinae Phalaris Phalaris Phalaris Phalaris Phalaris Phalaris Phalaris Phalaris Stipeae Stipeae Stipeae Miliinae Milieae Stipeae Miliinae Milium Milium Milium Milium Milium Milium Milium Zingeria Mibora Poeae Poeae Poeae Poeae Poeae Poeae Poeae Festucinae Festucinae Brizinae Brizinae Briza Briza Briza Briza Briza Briza Briza Briza Poinae Catabrosa Catabrosa Catabrosa Catabrosa Catabrosa Catabrosa September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1557

TABLE 1. Continued.

Ascherson and Graebner Clayton and Watson and (1898–1902) Maire et al. (1953) Prat (1960) Tzvelev (1976) Tutin et al. (1980) Renvoize (1986) Dallwitz (1992) Soreng et al. (2003)

Colpodium Colpodium Colpodium Koeleriinae Dissanthelium Apera Gymnachne Gymnachne Gymnachne Avellinia Avellinia Beckmannia Hellerochloa Hellerochloa Koeleria Mibora Parafestuca Parafestuca Cinninae Dissanthelium Cinna Cinna Scolochloeae Scolochloeae Scolochloinae Scolochloa Scolochloa Scolochloa Scolochloa Scolochloa Scolochloa Meliceae Meliceae Anthochloa Anthochloa Anthochloa Anthochloa Puccinellinae Catabrosa Catabrosa Pappophoreae Seslerieae Seslerieae Seslerieae Sesleriinae Sesleriinae Sesleriinae Oreochloa Oreochloa Oreochloa Oreochloa Oreochloa Oreochloa Oreochloa Sesleria Sesleria Sesleria Sesleria Sesleria Sesleria Sesleria Sesleria Ammochloinae Ammochloa Ammochloa Ammochloa Ammochloa Ammochloa Echinariinae Echinaria Echinaria Echinaria Echinaria Echinaria Echinaria

retrieved from GenBank and included in the analyses. Of these, 10 partially DeBry and Olmstead (2000), which consisted of random addition of taxa and complete ITS sequences (ITS1 and ITS2 spacers) were used in our TBR branch swapping, but saving fewer cladograms per replicate to reduce phylogenetic survey, and the absent characters (5.8S gene) were coded as computation time. missing data (Appendix). Gaps that were potentially informative were coded as Conflicts between the topologies obtained from Bayesian and parsimony binary presence/absence characters and added to the respective sequence data searches were analyzed visually. Incongruences between plastid and nuclear set for parsimony cladistic analysis. cladistic topologies were analyzed statistically using the nonparametric Bayesian and cladistic analyses were made on both individual and combined Wilcoxon signed rank test (Templeton, 1983; Mason-Gamer and Kellogg, plastid trnT-F and nuclear ITS data sets using the programs MrBayes v. 3.0 1996). For this purpose, separate trnT-F and ITS matrices of 92 common taxa (Huelsenbeck and Ronquist, 2002) and PAUP* v. 4.0 beta 10 (Swofford, were constructed from each data set, and a combined trnT-F/ITS matrix was 2002), respectively. Bayesian inference searches were performed independently also created. The trnT-F/ITS combination of data sets were compared by for each data set (trnT-F matrix, ITS matrix, and combined trnT-F þ ITS counting the number of steps required by each data set on its own optimal matrix) with MrBayes, using the optimal nucleotide substitution model topology (MP strict consensus) and on pairwise combinations with three previously selected for each case. This model was developed by calculating constraint topologies (the MP strict consensus and the 70% majority rule likelihood ratios for 56 substitution models using the program Model Test v. bootstrap consensus obtained from the rival data set and the MP strict 3.06 (Posada and Crandall, 1998). The three data sets generated the same consensus obtained from the combined data matrix). The number of steps optimal general time-reversible model with a proportion of invariable sites required in each case was calculated with PAUP* v. 4.0 beta 10, as indicated in (GTRþGþI) and four gamma rate categories, which was imposed on the Mason-Gamer and Kellogg (1996). Significance values of the Wilcoxon signed subsequent analyses. The analysis of each separate data set was performed rank tests were obtained from the Vassatstats online application (http:// through 1 000 000 generations by the Markov chain Monte Carlo (MCMC), faculty.vassar.edu/lowry/wilcoxon.html), which provide two-tailed significance with three hot chains (hot temperature 0.2) and one cold chain, sampling every values. 100 generations and allowing the program to estimate the likelihood parameters required. The log-likelihood scores of sample points were plotted against generation time to estimate the number of generations needed to converge to a RESULTS stable equilibrium value (Huelsenbeck and Ronquist, 2002; Leache´ and Reeder, 2002). Sampled points from the generation previous to stationary were The trnT-F data set—The sequenced plastid trnT-F region discarded using the burn-in option of MrBayes. New Bayesian searches of 5 000 000 MCMC generations were conducted for each data set using the comprised 2455 aligned nucleotide positions, 1195 of them topologies sampled from them to construct the respective 50% majority-rule corresponding to the trnT-L subregion and 1260 to the trnL-F consensus topologies and calculate the posterior probability support (PPS) of subregion. A total of 1065 positions were variable (43.3%, 496 their lineages. in trnL-F and 569 in trnT-L), and 586 were potentially Parsimony-based analyses were conducted through two heuristic searches, parsimony informative (23.8%, 252 in trnL-F and 334 in trnT- each aimed at recovering all possible equally shortest cladograms. An initial L). Informative gaps were frequent across the entire sequenced search was completed with the following parameters: closest addition of taxa, trnT-F region, and a 30-nucleotide gap (positions 355–384) tree-bisection-reconstruction (TBR) branch swapping, and the Mulpars option (multiple parsimony cladograms saved). The second search, which tried to find was shared by the members of the Koeleriinae core: Avellinia, other putatively shorter or equally parsimonious islands, consisted of 10 000 Gaudinia, Koeleria, Rostraria, Trisetum, and Parafestuca.By replicates of random-order-entry-starting cladograms with random addition of contrast, the trnL-F subregion had seven informative indels. A taxa, tree-bisection-reconnection (TBR) branch swapping, and saving no more large, 285-nucleotide gap (positions 219–503) was synapo- than 10 cladograms of length equal to or less than 10 per replicate. All equally morphic for the members of Koeleriinae (Avellinia, Gaudinia, parsimonious reconstructions obtained from the two searches were used to Graphephorum, Koeleria, Rostraria, Parafestuca, Sphenoph- compute the final strict consensus cladograms. Brachypodium distachyon (L.) P. Beauv. (Brachypodieae Harz) and Secale cereale L. (Triticeae) were used as olis,andTrisetum) and Aveninae (Arrhenatherum, Avena, and outgroups; B. distachyon was used to root the cladograms. Branch support for Helictotrichon subgenus Helictotrichon) lineages. Within the optimal topologies found under these parsimony strategies was estimated Koeleriinae, Gaudinia fragilis, Trisetum loeflingianum, and through 10 000 bootstrap replicates (Felsenstein, 1985) using the strategy of T. ovatum, here named the Trisetum ovatum group, showed a 1558 AMERICAN JOURNAL OF BOTANY [Vol. 94 common nine-nucleotide indel (positions 1000–1008), whereas Gaudinia, Parafestuca, and some representatives of annual Rostraria pumila, R. salzmannii, R. cristata, R. obtusiflora, Trisetum fell in the Koeleria s.l. subgroup, which included all Trisetum gracile, and T. flavescens, here named the Trisetum the Koeleria studied, whereas Avellinia and Rostraria were flavescens group, shared a large, 108-nucleotide gap (positions embedded in the Trisetum s.l. subgroup that encompassed the 897–1004). Another large gap of 188 nucleotides (positions remaining samples of Trisetum. Little resolution was observed 316–503) was synapomorphic for all representatives of Agro- in Koeleriinae, except for the strong relationships recovered for stidinae (Agrostis, Ammophila, Calamagrostis, Chaetopogon, the Trisetum ovatum group (Gaudinia fragilis, T. loeflingia- Gastridium, Polypogon, and Triplachne), as well as Briza, num, T. ovatum; 100% PPS, 96% BS), the Trisetum flavescens Airopsis, and Gymnachne. This group also shared an additional group (Rostraria spp., T. flavescens, T. gracile; 100% PPS, common five-nucleotide gap (positions 40–54). Holcus and 100% BS), and the Koeleria splendens/T. hispidum lineage Deschampsia s.s. (D. cespitosa, D. setacea) each had genus- (100% PPS; 86% BS). Agrostidinae had three early divergent specific gaps five nucleotides long (positions 235–239 and unresolved or poorly supported lineages (Gymnachne, Ammo- 164–168, respectively). phila, Calamagrostis) and a highly supported lineage (100% The Bayesian topologies reached a stable likelihood value BS) in which Triplachne/Gastridium (100% PPS; 97% BS) after the burn-in of 1000 phylograms. The 50% majority rule were sister to the Agrostis group (100% PPS) (that included the consensus phylogram is shown in Fig. 1. Phylogenetic sister Polypogon and Chaetopogon, 100% PPS, 97% BS). relationships are relatively well resolved at the deepest Briza joined with Airopsis in a highly supported group (100% branches of the trnT-F topology. In the parsimony-based PPS; 83% BS). analyses, the first heuristic search yielded 839 900 cladograms Poeae s.l. diverged in two weakly supported lineages: Poinae that were 2353 steps long (L) and had a consistency index (CI) plus several former Aveneae þ Puccinelliinae (78% PPS) and a (excluding uninformative characters) of 0.59 and retention large, polytomic, and poorly supported group that included index (RI) of 0.76. The second search did not find any further Loliinae and its close allies, Seslerieae, and the rest of the island of equally parsimonious cladograms. The strict consen- former Aveneae (89% PPS), such as Airinae, Deschampsia s.s., sus of all these most parsimonious cladograms (not shown) was Holcus, and Avenula s.s. The first lineage diverged into two compared with the Bayesian-based phylogram. Bayesian and groups: (1) a poorly supported group (55% PPS) with sister parsimony-based topologies were largely congruent and had relationships between Avenula pubescens and Milium, and similar levels of support for the main lineages (Fig. 1). The between and the closely related Anthochloa and supertribal Aveneae-Poeae-Seslerieae complex was resolved as Dissanthelium (100% PPS); (2) a group (100% PPS) monophyletic and highly supported (100% posterior probabil- comprising Cinna, Ventenata, and Alopecurus. Puccinelliinae ity support [PPS]; 100% bootstrap support [BS]) when Secale were formed by the sister taxa and Catabrosa cereale and Brachypodium distachyon were used as outgroups. (100% PPS; 100% BS). The highly supported Airinae (100% Within this complex, there was an early divergence between a PPS; 100% BS) comprised Aira, sister to a strong subgroup highly supported ‘‘core Aveneae’’ lineage (100% PPS; 100% composed of Corynephorus, Deschampsia maderensis, D. BS) and a Poeae s.l. lineage (100% PPS; 95% BS), the latter flexuosa, and Periballia (100% PPS; 89% BS). The second including the main Poeae subgroups plus many former lineage encompassed many former Aveneae and Poeae: Aveneae groups (Fig. 1). Core Aveneae comprised five highly Deschampsia s.s., Holcus, Seslerieae þ Mibora (98% PPS), supported subgroups: (1) Koeleriinae þ Aveninae þ Lagurus Parapholiinae/Cynosurinae (92% PPS), Dactylidinae þ Ammo- (100% PPS; 100% BS), (2) Anthoxanthiinae (100% PPS; chloa (89% PPS; 59% BS), and Loliinae þ Avenula (53% PPS). 100% BS), (3) Agrostidinae (100% PPS; 100% BS), (4) There was a close and strongly supported relationship of the Airopsis þ Briza (100% PPS; 83% BS), and (5) Phalaris. In the avenoid Dielsiochloa to Hellerochloa and to representatives of Bayesian phylogram, Phalaris was sister to a group formed by Festuca sect. Aulaxyper Dumort. (F. rubra group) (100% PPS; the other lineages, which further split into the sister subgroups 97% BS). Weakly supported sister relationship of Ammochloa (1)/(2) and (3)/(4), while Anthoxanthinae showed the sister to Dactylidinae (89% PPS) and the inclusion of Mibora in relationship of Anthoxanthum and Hierochloe; however, none Seslerieae (Sesleria, Oreochloa, and Echinaria), joined with of those relationships, except that of the sister lineages Oreochloa (100% PPS; 88% BS)were also evident. Agrostidinae/Airopsis þ Briza (99% PPS; 80% BS), were well supported (Fig. 1), and they collapsed into a polytomy in the ITS data set—The sequenced nuclear ITS region included parsimony-based topology (not shown). 665 aligned nucleotide positions, of which 400 were variable The Koeleriinae þ Aveninae þ Lagurus group had an initial (60.1%, 162 in ITS1 and 204 in ITS2) and 303 were potentially polytomy of Lagurus, Avena, and a series of remaining taxa parsimony informative (45.5%, 130 in ITS1 and 154 in ITS2). that diverged into successive weakly supported paraphyletic An eight-nucleotide gap in the ITS1 region (positions 55–62) Aveninae lineages (Helictotrichon s.s. and Arrhenatherum) and was considered synapomorphic for Koeleriinae (Gaudinia, a highly supported subgroup of Koeleriinae taxa (100% PPS; Koeleria, Graphephorum, Parafestuca, Rostraria, Sphenoph- 82% BS) (Fig. 1). In the parsimony-based cladogram, the three olis, and Trisetum). Aveninae genera with two species sampled were resolved as The Bayesian topologies reached a stable likelihood value monophyletic and sister to Koeleriinae but with low support after the burn-in of 1110 phylograms. The 50% majority rule (not shown). Within the Koeleriinae lineage, the American consensus phylogram is shown in Fig. 2. The first heuristic Graphephorum and Sphenopholis joined in a relatively well- search of our parsimony-based analyses yielded 510 800 supported sublineage (100% PPS; 72% BS), sister to an cladograms of L ¼ 2289, eight steps longer than the 25 equally Eurasian sublineage (parsimony-based cladogram) or collapsed shortest cladograms found by the second search (L ¼ 2281, CI with it (Bayesian phylogram). This group in turn diverged into ¼ 0.32, and RI ¼ 0.66). The strict consensus of all these most a highly supported Koeleria s.l. lineage (100% PPS; 90% BS) parsimonious cladograms (not shown) was compared with the and a less supported Trisetum s.l. lineage (96% PPS). Bayesian-based topology. Bayesian and parsimony-based September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1559

Fig. 1. The trnT-F search. The 50% majority rule consensus phylogram obtained by Bayesian inference. The scale represents 0.1 substitutions per site. Values above branches indicate posterior probability support (PPS)/bootstrap support (BS) values of the groups. A dash means BS , 50%. The groups that collapsed in the parsimony-based cladogram are marked with asterisks at their nodes. On the right, the black bar indicates those taxa formerly classified as Aveneae. 1560 AMERICAN JOURNAL OF BOTANY [Vol. 94

Fig. 2. ITS search. The 50% majority rule consensus phylogram obtained by Bayesian inference. The scale represents 0.1 substitutions per site. Values above branches indicate posterior probability support (PPS)/bootstrap support (BS) values of the groups. A dash means BS , 50%. The groups that collapsed in the parsimony-based cladogram are marked with asterisks at their nodes. On the right, the black bar indicates those taxa formerly classified as Aveneae. September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1561 topologies were largely congruent (Fig. 2), although the grouped with Milium and Phleum (65% PPS); (2) Apera þ relationships, especially at deep nodes, generally resolved Ventenata þ Alopecurus þ Beckmannia þ Cinna (91% PPS); better in the Bayesian topology. Despite the overall weakness (3) þ Avenula pubescens (100% PPS); (4) of the internal nodes of the ITS topology, the main lineages Colpodium þ Zingeria (100% PPS; 100% BS); and (5) recovered from the ITS data were largely congruent with those Puccinellinae (, Puccinellia,andCatabrosa; recovered from the trnT-F data (Fig. 1). In the ITS topology, 100% PPS; 98% BS). the supertribe Aveneae–Poeae–Seslerieae also was monophy- letic and well supported (100% PPS; 85% BS) when Combined ITS/trnT-F data set—Most of the conflicts Brachypodium, Secale, and Bromus were used as outgroups. affected relationships that were only weakly supported by one An early divergence separated Loliinae and allies (including set of data (Figs. 1 and 2). The main incongruences occurred in Dielsiochloa and Antinoria) (99% PPS; 67% BS) from all the distinct placements of the Seslerieae þ Mibora group in the remaining Aveneae–Poeae–Seslerieae taxa, which formed a trnT-F and ITS topologies. Sesleria, Oreochloa, Ammochloa, poorly supported group (52% PPS). Surprisingly, the avenoid and Mibora aligned within a highly supported Poeae s.l. Antinoria was sister to a well-supported Loliinae þ allies lineage in the plastid topology (Fig. 1) but within the poor to (100% BS; 86% BS) in which Parapholiinae/Cynosurinae moderately supported Aveneae þ Seslerieae þ Deschampsia þ (100% PPS; 99% BS) and Dactylidinae (100% PPS; 100% BS) Avenula s.s. lineage in nuclear topology (Fig. 2). The Wilcoxon lineages intermingled with Festuca. Dielsiochloa also was signed rank tests showed significant incongruence between the shown to be close to Festuca sect. Aulaxyper (F. rubra group), plastid and the nuclear topologies. The trnT-F data strongly joining in a highly supported subgroup with Hellerochloa rejected the ITS strict consensus (P , 0.001) and the 70% ITS (100% PPS; 86% BS). MR bootstrap consensus (P , 0.001); similarly, the ITS data The large group of the remaining taxa collapsed into three strongly rejected the trnT-F strict consensus (P , 0.001) and groups poorly supported by parsimony-based analyses. One of the 70% trnT-F MR bootstrap consensus (P , 0.001). these included the core Aveneae þ Seslerieae þ Scolochloa However, because of the scarce support recovered for many (100% PPS) and the Deschampsia s.s. þ Avenula s.s. þ of the relationships by the ITS data, their rejection by the trnT- Ammochloa (77% PPS) lineages, a second one the Airinae þ F data may not reflect strong conflict between them. The Holcus lineages (99% PPS), and the third one the Poinae þ combined topology, which is similar to the plastid one, was not allied lineages (100% PPS) (Fig. 2). Weak divergences and rejected by the plastid trnL-F-region data subset (P . 0.26) but polytomies were found at the deep nodes of the core Aveneae þ was weakly rejected by the plastid trnT-L-region data subset Seslerieaeþ Scolochloa group. Koeleriinae (including Lagurus) (0.036 , P , 0.073) and by the ITS data set (0.030 , P , (100% PPS) and Aveninae (including Seslerieae) (65% PPS) 0.060), thus indicating some discordances within the plastid formed two groups, but the second was weakly supported. data set. While this test is useful for global comparisons, it is Within the largely unresolved Koeleriinae, the Trisetum also recognized to be problematic; topologies are likely to be flavescens group again was recovered (100% PPS), with rejected topologies because of the presence of spurious nodes Rostraria subsp. (99% PPS, but excluding Rostraria cristata, in the rival constraint (Mason-Gamer and Kellogg, 1996). In R. hispida, and R. obtusiflora) clearly separated from perennial addition, the overall high congruence between the plastid and Trisetum (96% PPS). Within the Trisetum ovatum group, only nuclear Aveneae and Poeae lineages moved us to combine the T. duforei and T. loeflingianum (100% PPS) had any two matrices and to perform further phylogenetic analyses. The relationship. Koeleria pyramidata and K. dasyphylla were combined trnT-F/ITS data set was made of 92 co-sequenced linked (99% PPS). Aveninae resolved into two separate accessions (Appendix). In the combined topologies, it was lineages: a relatively well-supported Avena group (94% PPS; clear that the large, highly structured, plastid DNA data set 77% BS) with sectional resolution as reported by Rodionov et overwhelmed the much smaller and less informative ITS data al. (2005), that formed a polytomy with the Seslerieae set. Because of the scant information provided by the combined representatives plus Mibora. In turn, Arrhenatherum, Helicto- topology, we did not show it here, referring to it only when trichon, and Pseudarrhenatherum formed a group (93% PPS) justified. in which Helictotrichon was paraphyletic to a well-supported subgroup (100% PPS) that also included Pseudarrhenatherum. Within the better-sampled and strongly supported Anthoxan- DISCUSSION thinae (100% PPS; 100% BS), a polytomy is formed by Hierochloe and Anthoxanthum lineages in the Bayesian Aveneae core lineage—Although it had been suggested that phylogram (Fig. 2). Agrostidinae formed a moderately Aveneae were more advanced than Poeae (Clayton and supported lineage (96% PPS), except for Calamagrostis and Renvoize, 1986), our analyses confirmed the insights of Ammophila, which joined with Airopsis (54% PPS), and Soreng and Davis (2000), which indicated that Aveneae and Agrostis was paraphyletic and most of its sampled species Poeae were intermingled. Despite this admixture, our com- formed a group in which Chaetopogon was embedded (100% bined and plastid analyses recovered two main groups PPS; 93% BS). A strong relationship between Triplachne and composed of mainly Aveneae and mainly Poeae taxa, Gastridium was again recovered by these data (100% PPS; respectively (nuclear analyses did not render these two quite 100% BS). Phalaris and Briza, collapsing at the base of the definited groups but this should be carefully considered due to group, and Scolochloa, as sister to the whole group, completed the scarce support obtained for this topology at its deepest this weak and large lineage. The Airinae lineage included nodes). The group primarily with Aveneae is named hereafter Deschampsia flexuosa, D. maderensis, Periballia, and Cor- the core Aveneae lineage, because it included representatives ynephorus (98% PPS; 60% BS). Poinae þ Puccinellinae (100% of the most typical infratribal Aveneae taxa (Table 1), PPS) had a large basal polytomy of five lineages: (1) Poa þ Koeleriinae, Aveninae, and Agrostidinae, as well as minor Anthochloa/Dissanthelium (94% PPS), which were weakly groups obscurely related to the previous groups, such as 1562 AMERICAN JOURNAL OF BOTANY [Vol. 94

Lagurus, Anthoxanthinae, Airopsis, Phalaris, and Scolochloa only by plastid data (Fig. 1). This Trisetum flavescens group, (Figs. 1 and 2). Additionally, it included a few Poeae enlarged with Rostraria, corresponds with Trisetum sect. representatives such as Briza, Parafestuca, and Gymnachne Trisetum. This section was typified by T. flavescens and (Figs. 1 and 2), although some Poeae representatives characterized by more or less open, oval-to-pyramidal panicles potentially related to this core Aveneae, such as Chascolytrum, (Finot et al., 2004, 2005b). The close relationship found Poidium,andTorreyochloa, were not studied here (cf. Soreng between Trisetum and Rostraria agrees with earlier reports that and Davis, 2000). highlighted the strong resemblances between the lemma of both genera (Hubbard, 1937; Holub, 1974; Jonsell, 1978). Koeleriinae and Lagurus—Trisetum has been considered the Rostraria cristata, R. obtusiflora, R. hispida, and T. paniceum ancestral lineage of Koeleriinae because of its less reduced were resolved by ITS data as relatives of the T. ovatum group, lemma (Clayton and Renvoize, 1986; Mosulishvili, 2000). The but with little support (Fig. 2). Another Trisetum group, formed lemma of Trisetum is three-awned with a main, often by T. baregense, T. glaciale, and T. paniceum, was recovered geniculate, dorsal awn inserted from low down to near the only by the combined data set (not shown) but not by separate top, and two more or less developed, additional awnlets arising plastid and nuclear data. Trisetum glaciale was resolved as a from the lateral veins at the apex. The closely related perennial sister to the T. baregense–T. spicatum group in a moderately Koeleria has muticous, mucronate, or apically or subapically supported lineage (Fig. 2). This group corresponds with awned lemma. Trisetum sect. Trisetaera Asch. & Graebn., typified by T. Koeleriinae also includes ephemeral species adapted to dry, spicatum and characterized by dense, spiciform, and narrow open places from the Mediterranean to the western Himalayas. panicles (Finot et al., 2004, 2005b). Avellinia, only studied for They are placed either in Trisetaria Forssk., a name lately plastid data, remained in an unresolved placement within this fallen into disuse and applied to the annual species of Trisetum, last group (Fig. 1). This genus might be an independent annual or in Rostraria, a small genus with straight, or sometimes derivation from a different perennial Trisetum line. slightly curved, subapical awned lemmas. Additionally, the Graphephorum and Sphenopholis were placed within annual Mediterranean genus Avellinia was placed in Trisetaria Koeleriinae by all analyses (Figs. 1 and 2) and resolved as (Clayton and Renvoize, 1986) or in Rostraria (Romero Zarco, sister lineages by the plastid and combined topologies (Fig. 1). 1996) because of similarities in its lemma. The American These results confirm the close relationships found between Sphenopholis, separated from Trisetum by Scribner (1906) Sphenopholis and Trisetum by Soreng and Davis (2000) and because of its ovate glumes and florets disarticulating below the morphologic affinities between Graphephorum and Trise- the glumes, and Graphephorum, with a lemma with an entire tum reported by Finot et al. (2005a). However, a larger apex and a reduced dorsal awn (subapical mucro) (Finot et al., sampling of American Koeleria and Trisetum taxa is needed 2004, 2005a, b), were also considered to be related to Trisetum before their systematics can be further assessed. Finally, this (Clayton and Renvoize, 1986). Koeleriinae lineage can be distinguished by a set of Koeleriinae were recovered in all our analyses, although they morphologic features, such as 2–5 florets per spikelet, a were more strongly supported by the plastid data (cf. Figs. 1 relatively small spikelet, a keeled lemma (as opposed to more and 2). Neither Koeleria nor Trisetum formed monophyletic or less rounded lemmas, as in Avellinia, Gaudinia, and some groups, but all Koeleria representatives were grouped into a Trisetum species), a poorly developed awn, glabrous ovary well-supported lineage, which also included several species of (hairy in Gaudinia and in some Trisetum species, with an Trisetum. Relationships within Koeleria spp. were not resolved apical appendage in Graphephorum), short hilum, and liquid satisfactorily by the present analyses, and K. vallesiana and K. endosperm. crassipes appeared to be polyphyletic. Parafestuca, a mono- The Mediterranean genus Lagurus was part of a polytomy typic endemic genus from Madeira traditionally included in the that included either the Koeleriinae and Aveninae lineages by Poeae (Alexeev, 1985; Clayton and Renvoize, 1986; Watson plastid data (Fig. 1) or only Koeleriinae representatives by and Dallwitz, 1992), was placed within this group (Figs. 1 and nuclear data (Fig. 2). Lagurus has historically been included in 2). Parafestuca was included in Koeleria, not only because of Agrostidinae (Table 1) based on its one-flowered spikelets, but its placement here, but also because its morphology is typical other general features of this monotypic genus, such as a of the genus Koeleria (Soreng et al., 2003; Quintanar et al., glabrous ovary, short hilum, and liquid endosperm, connect it 2006). This mostly perennial lineage also included the to Koeleriinae. Trisetum ovatum group, that comprised the annual T. loeflingianum, T. ovatum, and the small genus Gaudinia,with Aveninae—Genera of this subtribe have been characterized either strongly contracted panicles (Trisetum representatives) or traditionally by their long glumes, laterally compressed racemes of spikelets (Gaudinia). However, the circumscription spikelets with 1–2 (7) female fertile florets, and more or less of this group was less clear in the ITS topology, which developed dorsal awns. Helictotrichon s.s. (excluding Avenula, recovered only a strong relationship for T. loeflingianum and T. discussed later), traditionally considered as perennial oats, duforei (Fig. 2). Another Trisetum species, the perennial T. separates from the mostly annual Avena based on its scabrid hispidum, was unexpectedly linked with Koeleria splendens in and more strongly keeled glumes and on its linear-lanceolate a relatively well-supported group (Figs. 1 and 2). lodicules (Baum, 1968); however, the taxonomic boundaries The remaining Trisetum taxa were included in a weakly between Helictotrichon and other perennial Aveninae, such as supported group that also included all the Rostraria taxa the European and Mediterranean Arrhenatherum with an either studied and Avellinia (Fig. 1). The relationships of R. pumila hermaphroditic or female upper floret, and the Western and R. salzmannii (plus R. litorea, Fig. 2) with the perennial T. European Pseudarrhenatherum, with one incomplete floret flavescens and T. gracile (plus T. turcicum in Fig. 2) were proximal to the hermaphroditic one, were never clear on the corroborated by all the data sets, whereas the relationships of R. basis of morphology. cristata and R. obtusiflora with this last group were supported Aveninae, including Avena, Arrhenatherum, Pseudarrhena- September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1563 therum,andHelictotrichon, were paraphyletic to Koeleriinae in thum and Hierochloe also share aromatic coumarin-scented the plastid Bayesian phylogram (Fig. 1) but were united in a shoots, glabrous ovary, short hilum, small embryo, and hard weakly supported group sister to Koeleriinae in the parsimony- endosperm, whereas Phalaris differs because of its non- based topologies and nuclear phylogram (Fig. 2), suggesting a aromatic shoots, small or large embryo, and a long-linear potential origin of Koeleriinae from within Aveninae. Helicto- hilum. The first two genera joined in a strongly supported trichon, including most of the sampled taxa of this genus group sister to Koeleriinae þ Aveninae lineages (Figs. 1 and 2), (subgenus Helictotrichon), and Pseudarrhenatherum were and nuclear data additionally showed a paraphyletic perennial grouped, agreeing with the limited morphologic differentiation Hierochloe including a perennial-to-annual monophyletic found between them (Clayton and Renvoize, 1986). Helicto- Anthoxanthum in the parsimony-based topology (not shown). trichon subgenus Tricholemma Ro¨ser (H. jahandiezii), on the Anthoxanthinae included just these two genera; despite the other hand, was sister to Arrhenatherum (Fig. 2). This relationship between Anthoxanthum and Phalaris found by Aveninae lineage can be characterized by a set of morphologic Soreng and Davis (2000), our data clearly separated them from features such as large spikelets, 1–7 florets per spikelet, hairy Phalaris (Figs. 1 and 2). Phalaridiinae is restricted to Phalaris, ovary, long-linear hilum, grooved embryo, and mostly solid a genus of still uncertain relationships with respect to the endosperm. groups mentioned earlier, although the plastid topology suggests that Phalaris is sister to all other core Aveneae Agrostidinae, Anthoxanthinae, and allies—Agrostidinae, the (Fig. 1). third main subtribe of Aveneae, have been characterized On the other hand, Scolochloa, a wet-meadow grass from the mainly by their small, one-flowered spikelets. They were northern hemisphere that was classified in its own tribe resolved as one of the main lineages of the core Aveneae (Figs. Scolochloeae Tzvelev or as a subtribe of Poeae (Table 1), was 1 and 2). The large genus Agrostis was resolved as para/ sister to all core Aveneae lineages in the ITS phylogram (Fig. polyphyletic in the ITS analysis, with Chaetopogon nested in a 2, not sampled in the plastid analyses). Its hairy ovary, long- strongly supported Agrostis s.s. subgroup sister to A. truncatula linear hilum, and hard endosperm may be plesiomorphic in (Fig. 2). A. truncatula differs from other Agrostis in the core Aveneae (cf. Baum, 1968). microstructure of the lemma and in its open and diffuse panicle and was placed in subgenus Zingrostis by Romero Garcı´a et al. Former Aveneae lineages related to the traditional Poeae (1988). The strong sister relationship recovered for the pan- subtribes—Loliinae and its allied subtribes Parapholiinae, Mediterranean genera Triplachne and Gastridium in all Cynosurinae, and Dactylidinae, and Poinae and its allied analyses corroborate previous ideas about their morphologic subtribe Puccinelliinae had poorly supported relationships to affinities (Clayton and Renvoize, 1986). Calamagrostis and many former Aveneae groups, and/or incorporated other genera Ammophila were placed in Agrostidinae (Fig. 1), but their also classified as Aveneae (see Dielsiochloa and Loliinae) position was unresolved using nuclear data (Fig. 2). All these (Figs. 1 and 2). Seslerieae were close to Aveninae (ITS, Fig. 2) genera have been included in Aveneae, except Gymnachne. or sister to Parapholiinae/Cynosurinae (trnT-F, Fig. 1), or to the This South American genus has many florets (3–6) per spikelet whole large Poeae þ former Aveneae group in the combined and one stamen per floret and has been placed in Poeae (Table topologies. 1) or in Poeae subtribe Brizinae (Soreng et al., 2003). This Agrostidinae lineage has a set of morphologic features, such as Closest relatives of Poinae—A morphologically diverse small spikelets, a single floret per spikelet (except Gym- assemblage of Aveneae, such as Avenula pubescens, Alopecu- nachne), glabrous ovary, short hilum (long-linear in Ammo- rus, Apera, Beckmannia, Cinna, and Ventenata, formed a well- phila), and mostly solid endosperm (liquid in some Agrostis supported group with Poa and relatives, although relationships and Polypogon species), that in conjunction separate Agros- within this large group were unclear (Figs. 1 and 2). Avenula tidinae from both Koeleriinae and Aveninae. pubescens represents an independent split from Helictotrichon Eurasian Briza traditionally have been classified as Poeae in (Helictotrichon subgenus Pubavenastrum (Vierh.) Holub), and all morphology-based systems (Table 1) but were included it was not related to either the core Aveneae-allied Helicto- within Aveneae in molecular surveys (Soreng et al., 1990; trichon s.s. or the Loliinae-allied Avenula s.s. (discussed later). Hsiao et al., 1995; Soreng and Davis, 2000; see also Figs. 1 and This species is morpho-anatomically quite different from other 2). The small subapical awns in Briza and other closely related Avenula (Romero Zarco, 1984; Ro¨ser, 1989, 1997), although Poeae, such as Chascolytrum and Poidium, were considered to its external appearance is that of a ‘‘typical’’ Aveninae. be a morphologic evidence of their affinities to Aveneae Grebenstein et al. (1998) and Soreng and Davis (2000; plastid (Soreng and Davis, 2000). The small genus Airopsis, which data alone) also recovered an isolated placement for this plant, was classified historically as Airinae (Maire et al., 1953; Albers close to Alopecurus (Grebenstein et al., 1998; BS 75%). Here, and Butzin, 1977) based on its putative similarity with Aira, A. pubescens joined with Arctagrostis as part of a polytomy in was placed with other representatives of the subtribe in the a poorly supported ITS-based group (Fig. 2). This latter genus, vicinity of Agrostidinae and Briza. The broadly ovate, which has sometimes been placed in Aveneae (Table 1), was ventricose glumes of monotypic Airopsis resemble those of shown to be a close ally of Poa subg. Andinae in the more Briza, and this may reflect its true affinities. The plastid data detailed study of Poinae of Gillespie et al. (2006). showed a sister relationship of Briza plus Airopsis to Agro- In the same assemblage we found Alopecurus and Phleum, stidinae, but this relationship was not supported by nuclear data two genera distributed throughout the temperate northern (Figs. 1 and 2). hemisphere and South America and traditionally placed in Anthoxanthum, Hierochloe,andPhalaris, traditional mem- Aveneae (Table 1) (Figs. 1 and 2), confirming previous bers of Anthoxanthinae (Table 1), have one distal female-fertile findings by Soreng and Davis (2000) and Gillespie et al. floret per spikelet with proximal sterile—Anthoxanthum, (2006). Beckmannia, characterized by its long glumes and one- Phalaris—or male/neuter—Hierochloe—florets. Anthoxan- or two-flowered spikelets and usually treated as Aveneae 1564 AMERICAN JOURNAL OF BOTANY [Vol. 94

(Table 1), has also been considered to be related to Poeae traditionally divided into two sections that were later (Avdulov, 1931; Reeder, 1953) and was placed close to segregated into two independent genera, Deschampsia and Alopecurus in the ITS topology of Rodionov et al. (2005; BS Avenella, based on differences in lodicule shape, root 66%). The European-Mediterranean Apera, commonly includ- histology, and spikelet architecture (Frey, 1999). The recent ed in Agrostidinae (Table 1) because of its single-flowered phylogenetic study of Chiapella (2007), based on combined spikelet, was also placed in Poeae (Tutin et al., 1980). The nuclear and plastid data, has shown independent origins for temperate Eurasian and American Cinna (Aveneae or Aveni- these two genera, a scenario also corroborated by our data nae, see Table 1), was resolved as a close relative of (Figs. 1 and 2). Airinae, excluding Deschampsia s.s., showed Sphenopholis and Trisetum by Soreng and Davis (2000). Its the successive early divergences of Aira and Corynephorus and alignment with Poinae and relatives is consistent with its included Avenella, represented here by the European De- recognition as subtribe Cinninae Caruel of Poeae. The schampsia flexuosa and the Madeiran endemic D. maderensis unexpected placement recovered for the small xerophytic (Fig. 1). Plastid data suggest that Avenella is paraphyletic, the annual genus Ventenata contradicts traditional classifications in Mediterranean annual Periballia being derived from within it which it was included in Avena (Reichenbach, 1830; Koch, (Fig. 1). Despite the suggested affinities of Holcus and 1854; Ledebour, 1853), Aveneae, or the Trisetum group Deschampsia (Clayton and Renvoize, 1986), our study did (Clayton and Renvoize, 1986) (Table 1). The nongaping paleas not confirm a close relationship of Holcus to either and slightly grooved caryopsis (Eig, 1929) separate Ventenata Deschampsia s.s. or Airinae, it being weakly resolved as sister from Koeleriinae, and the phylogenetic relationships recovered group to this last lineage only in nuclear analyses (Fig. 2). The here could support its classification in a separate subtribe, morphologic distinctness of Holcus, which is characterized by Ventenatinae. its modified spikelets with a lower, awnless hermaphroditic Two small Eurasian genera, Colpodium and Zingeria, floret, an upper, straight-to-hooked, awned male floret, and formed a strongly supported lineage in this assemblage (Fig. variable base chromosome number (n ¼ 4, 7), supports its 2). Despite previous attributions of Zingeria to Agrostidinae or placement in the monogeneric subtribe Holcinae. The place- Aveneae (Table 1), this group was also reported by Rodionov ments of Deschampsia s.s., Airinae, and Holcus, being part of a et al. (2005; BS 100%) in a sister placement to Alopecurus and polytomy with Loliinae and relatives (Deschampsia and Beckmannia (BS 52%). Both genera have a single-flowered Holcus) or sister to them (Airinae) (Fig. 1), is not supported spikelet, more or less awnless lemma, glabrous ovary, short by the nuclear data, that placed them sister to core Aveneae hilum, and a reduced base chromosome number, from 2 to 4. (Deschampsia plus Avenula) or in polytomy with all of them The paleoartic genus Milium, with dorsiventrally compressed, and Poinae and its relatives (Airinae and Holcus) (Fig. 2). one-flowered spikelets and awnless lemma, has been placed in The separation of Helictotrichon s.s. (see Aveninae) from either Aveneae, Stipeae Dumort., or its own tribe, Milieae Link Avenula (Helictotrichon subgenera Pratavenastrum (Vierh.) (Table 1). The position of Milium in our topologies confirms Holub) has been controversial because of their general the results of Soreng and Davis (2000) and Gillespie et al. similarity to Aveninae, although other morpho-anatomic (2006), definitively places it in this heterogeneous group (Figs. characters distinguish them (Kergue´len, 1975; Tutin et al., 1 and 2), and is consistent with its treatment as a separate 1980; Gervais, 1983). Our plastid analyses resolved Avenula subtribe. Finally, despite the placement of Anthochloa in s.s. as sister lineage to Loliinae (Fig. 1), and including Aveneae (Stebbins and Crampton, 1961) or Meliceae (Clayton Ammochloa, a small annual Mediterranean genus usually and Renvoize, 1986), this monotypic genus, characterized by a treated as Poeae or Seslerieae (Table 1) and, more rarely, as broadly expanded, flabellate lemma, and the pooid Dissanthe- sister to Aveneae (MacFarlane, 1987). Nuclear data placed lium (Aveneae in Clayton and Renvoize, 1986), were found to Avenula sister to Deschampsia s.s. (Fig. 2), agreeing with be sister to Poa, although with rather poor support (Figs. 1 and Grebenstein et al. (1998; 40% BS). This placement for Avenula 2). This confirms their previous placement in Poeae (Soreng et separate from the Aveneae core lineages agrees with the earlier al., 2003) and findings by Gillespie et al. (2006) that places findings of Grebenstein et al. (1998) and Soreng and Davis these and other minor genera such as Austrofestuca and (2000) and corroborates the polyphyly of Helictotrichon s.l. Eremopoa with Poa. The plastid data placed Ammochloa as sister to Dactylidinae Puccinelliinae were weakly supported as sister to Poinae and but with poor support (Fig. 1). The main morphologic features relatives by the plastid data alone (Fig. 1) and included of Ammochloa, a condensed , glabrous ovary, Puccinellia, Catabrosa, and Sclerochloa. The close relation- short hilum and hard endosperm, are not those of Avenula but ship of the holarctic Puccinellia and the pan-Mediterranean are shared by the closest relatives of Loliinae (Parapholiinae, Sclerochloa in the nuclear topologies confirms earlier findings Cynosurinae, and Dactylidinae). However, the high mutation by Choo et al. (1994) and Catala´n et al. (2004). The linking of rate of the trnT-F region of Ammochloa, reflected by its long the helophytic to mesophytic Catabrosa, included in Meliceae branch (Fig. 1), may have disturbed the reconstruction of the (Watson and Dallwitz, 1992), to this group also supports plastid phylogeny. previous findings (Soreng et al., 1990; Choo et al., 1994; Festuca sect. Aulaxyper (F. rubra group) showed a strongly Gillespie et al., 2006). All these genera have (1) 2–10 florets supported sister relationship to a lineage formed by Dielsio- per spikelet, noncarinate lemma, glabrous ovary, short hilum, chloa and the festucoid Hellerochloa, two restricted Central and hard endosperm. and South American genera (Figs. 1 and 2). Dielsiochloa,a small endemic genus restricted to and , tradition- Closest relatives of Loliinae—Airinae, which traditionally ally has been included in Aveneae (Table 1). Despite its long- included the large, worldwide Deschampsia, Aira, and several linear hilum and hard endosperm, it has been considered to be other small genera, are characterized by 2 (3) florets per related to Trisetum because its lemmas have straight dorsal spikelet and usually straight or somewhat bent awns originating awns (Clayton and Renvoize, 1986). Finally, Antinoria, a small from near the base to below the lemma apex. Deschampsia was Mediterranean genus with short hilum, glabrous ovary, and September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1565 characteristic of damp places, was considered as part of Airinae origin of each group and the nature of potential horizontal gene by some authors (Albers and Butzin, 1977). Our results placed transfer events, thus unraveling the evolutionary history of the it as a sister group to Loliinae, Parapholiinae, Cynosurinae, and supertribal complex. Dactylidinae (Fig. 2). Large, species-rich lineages, such as Koeleriinae, Agro- stidinae, Loliinae, Poinae, etc., with a strong internal structure Misplaced lineages: Seslerieae and Mibora—The strongest were generally distinguished from other small, less-diversified, conflicts observed between plastid and nuclear topologies satellite lineages, such as Airinae, Alopecurus, Anthoxanthi- affected the placements of the representatives of Seslerieae and nae, Antinoria, Avenula, Briza, Deschampsia, Holcus, Milium, Mibora. Sesleria, Oreochloa, and Echinaria, three European Phalaris, Phleum, and Scolochloa. Most of them are usually and Mediterranean genera characterized by their strongly sister groups to these large lineages groups in our topologies condensed inflorescences and traditionally classified in Sesler- (Figs. 1 and 2). The relatively high frequency of annual ieae (Table 1), along with Mibora, formed a moderately radiations of species adapted to xeric environments in the large supported group in both analyses (Figs. 1 and 2). Seslerieae groups contrasts with the predominant mesophytic-to-helo- and Mibora linked with core Aveneae or with Poeae in the phytic perennial elements of the satellite groups. Soreng and nuclear and plastid topologies, respectively. This could indicate Davis (2000) speculated on the potential ancestry of the a hybrid origin of the representatives of this lineage. helophytic Amphibromus, Torreyochloa, and Scolochloa, also Morphology does not confirm the unexpectedly strong first divergent elements in their topologies, thus indicating a relationship recovered between Mibora and Oreochloa using temperate wetland origin for Aveneae and Poeae. Our results plastid data (Fig. 1). The western Mediterranean–Atlantic seem to support that hypothesis, but some of those satellite genus Mibora, characterized by its dwarf habit and single- groups do not retain some of the characters considered flowered spikelets, traditionally has been placed in either ancestral of the complex, i.e., long-linear hilum, hairy ovary, Agrostidinae (sometimes in its own subtribe Miborinae Asch. multiflowered spikelets, multinerved glumes, and elaborate & Graebn.) or Alopecurinae (Table 1). Soreng and Davis awns (cf. Baum, 1968). This reinforces the idea of a widely (2000) found Mibora resolved in Agrostidinae in their cladistic extended morphologic homoplasy within the complex. analysis of structural data, but plastid data alone placed it in Poeae; Soreng et al. (2003) tentatively placed it in Miliinae. Taxonomic recommendations—Systematically, the neat The high mutation rate of this annual lineage, reflected by its separation of the Aveneae core lineages (Aveninae, Agros- long branches (Figs. 1 and 2), could have disturbed the parsimony and Bayesian reconstructions. Further analyses of tidinae, Koeleriinae, Anthoxanthinae, and allies) from Poeae larger samples of the Seslerieae group representatives are and the remaining former Aveneae groups in the plastid required to clarify their relationships. topology could support tribal status for the groups that form that core. Certainly, acceptance of this tribal rank would be Evolutionary history of Aveneae, Poeae, and Seslerieae— consistent with most traditional classificatory proposals in The presence of many groups, formerly placed in Aveneae, that Pooideae (Table 1). However, many former Aveneae lineages our analyses place in the neighborhood of Loliinae or Poinae mentioned should be excluded from it, and the reconstituted could be due to past hybridizations. Seslerieae, which have Aveneae would lack its distinctive morphologic features. An pooid plastid and avenoid nuclear genomes, may have resulted alternative treatment could be the division of the complex into from past intertribal reticulation events that resulted in a new, three subtribes: (1) the Aveneae core groups, (2) Poinae and its morphologically distinct lineage. In fact, extensive past closest Aveneae relatives, and (3) Loliinae and its closest reticulation has been repeatedly invoked to explain the failure Aveneae relatives. However, these groups have overall weak to reconcile topologies recovered from nuclear and plastid data support, especially by nuclear data, and they also lack in Pooideae (Davis and Soreng, 1993; Mason-Gamer and distinctive features. In accordance with the proposals of Soreng Kellogg, 1996) and to explain the presence of pooid taxa with and Davis (2000) and other authors (Tzvelev, 1989, pro parte; an avenoid genome and vice versa (Soreng and Davis, 2000). GPWG, 2001; Soreng et al., 2003), we propose to accept an Although our nuclear ITS data do not strongly contradict our enlarged Poeae that would include the former Aveneae, Poeae, plastid data, some former Aveneae, such as Deschampsia s.s. and Seslerieae lineages. This tribe can be split into different and Avenula s.s., were placed sister to core Avenae by the subtribes, such as Aveninae, Koeleriinae, Agrostidinae, nuclear data (Fig. 2), despite the more distant relationship Loliinae, Poinae, and others, as we probe its phylogenetic provided by combined and plastid data (Fig. 1). This could structure more deeply. This way we can avoid taxonomic suggest potential topologic disturbances caused by the putative conflicts between the phylogeny of these lineages and the paralogy of ITS ribotypes and homoplasy, a phenomenon that maintenance of older ranks that lack the morphologic attributes often blurs ITS phylogenetic reconstructions, especially in that define them. polyploid-rich groups (A´ lvarez and Wendel, 2003; Stace, 2005) such as some Aveneae. This scenario also could indicate LITERATURE CITED that the morphologic traits of many former Aveneae lineages could be plesiomorphic or largely homoplasious. It also might ALBERS, F., AND F. BUTZIN. 1977. Taxonomie und Nomenklatur der reflect the consequences of lineage sorting if the ancestral Subtriben Aristaveninae und Airinae (Gramineae–Aveneae). Will- denowia 8: 81–84. Aveneae, Poeae, and Seslerieae diversified faster than the ALEXEEV, E. B. 1985. Novye rody slakov. Byulleten’ Moskovskogo fixation of gene copies in these lineages. However, reticulation Obshchestva Ispytatelej Prirody. Otdel biologicheskii 90: 102–109. and lineage sorting scenarios are not easily differentiated a A´ LVAREZ, I., AND J. F. WENDEL. 2003. Ribosomal ITS sequences and plant priori (Kellogg et al., 1996), and both could have operated phylogenetic inference. Molecular Phylogenetics and Evolution 29: together (Catala´n et al., 2004). Further analysis of other nuclear 417–434. single-copy genes and organellar genes might help to detect the ASCHERSON,P.F.A.,AND K. O. P. P. GRAEBNER. 1898–1902. Synopsis der 1566 AMERICAN JOURNAL OF BOTANY [Vol. 94

Mitteleuropaischen Flora, vol. Glumiflorae 1. Graminae. Wilhelm the Festuca–Lolium complex (Poaceae) based on ITS sequence data. Engelmann, Leipzig, . 795 pp. Plant Systematics and Evolution 224: 33–53. AVDULOV, N. P. 1931. Karyo-systematiche untersuchung der familie GERVAIS, C. 1983. Wide hybridization attempts in the tribe Aveneae Nees. Gramineen. Trudy po prikladnoi botanike, genetike i selektsii. Botanica Helvetica. Bulletin de la Socie´te´ Botanique Suisse. 93: 195– Supplement 44. 212. BALDWIN, B. G., M. J. SANDERSON,J.M.PORTER,M.F.WOJCIECHOWSKI,C. GILLESPIE, L. J., A. ARCHAMBAULT, AND R. J. SORENG. 2006. Phylogeny of S. CAMPBELL, AND M. J. DONOGHUE. 1995. The ITS region of nuclear Poa (Poaceae) based on trnT—trnF sequence data: major clades and ribosomal DNA: a valuable source of evidence on angiosperm basal relationships. Aliso 23: 420–434. phylogeny. Annals of the Missouri Botanical Garden 82: 247–277. GPWG (GRASS PHYLOGENY WORKING GROUP). 2001. Phylogeny and BAUM, B. R. 1968. Delimitation of the genus Avena (Gramineae). subfamilial classification of the grasses (Poaceae). Annals of the Canadian Journal of Botany 46: 121–132. Missouri Botanical Garden 88: 373–457. BLATTNER, F. R. 2004. Phylogenetic analysis of Hordeum (Poaeae) as GREBENSTEIN, B., M. RO¨ SER,M.SAUER, AND V. HEMBELEN. 1998. Molecular inferred by nuclear rDNA ITS sequences. Molecular Phylogenetics phylogenetic relationships in Aveneae (Poaceae) species and other and Evolution 33: 289–299. grasses as inferred from ITS1 and ITS2 rDNA sequences. Plant BRYSTING, A. K., M. F. FAY,I.J.LEITCH, AND S. G. AIREN. 2004. One or Systematics and Evolution 213: 233–250. more species in the arctic grass genus Dupontia?—a contribution to HOLUB, J. 1974. New names in Phanerogamae 3. Folia geobotanica et the Panarctic Flora project. Taxon 53: 365–382. phytotaxonomica 9: 261–275. CATALA´ N, P., E. A. KELLOGG, AND R. G. OLMSTEAD. 1997. Phylogeny of HSIAO, C., N. J. CHATTERTON, AND K. H. ASAY. 1995. Molecular phylogeny Poaceae subfamily Pooideae based on chloroplast ndhFgene of the Pooideae (Poaceae) based on nuclear rDNA (ITS) sequences. sequences. Molecular Phylogenetics and Evolution 8: 150–166. Theoretical and Applied Genetics 90: 389–398. CATALA´ N, P., P. TORRECILLA,J.A.LO´ PEZ RODRI´GUEZ, AND R. OLMSTEAD. HUBBARD, C. E. 1937. Trisetum. In A. W. Hill [ed.], Flora of tropical 2004. Phylogeny of the festucoid grasses of subtribe Loliinae and . L. Reeve, Kent, UK. 192 pp. allies (Poeae, Pooideae) inferred from ITS and trnL-F sequences. HUELSENBECK, J. P., AND F. RONQUIST. 2002. MrBayes. Bayesian inference Molecular Phylogenetics and Evolution 31: 517–541. of phylogeny. v. 3.0. Bioinformatics 17: 754–755. CHARMET, G., C. RAVEL, AND F. BALFOURIER. 1997. Phylogenetic analysis in HUNTER, A. M., D. A. ORLOVICH,K.M.LLOYD,W.G.LEE, AND D. J. the Festuca-Lolium complex using molecular markers and ITS rDNA. MURPHY. 2004. The generic position of Austrofestuca littoralis and the Theoretical and Applied Genetics 94: 1038–1046. reinstatement of Hookerochloa and Festucella (Poaceae) based on CHIAPELLA, J. 2007. A molecular phylogenetic study of Deschampsia evidence from nuclear (ITS) and chloroplast (trnL-trnF) DNA (Poaceae: Aveneae) inferred from nuclear ITS and plastid trnL sequences. New Zealand Journal of Botany 42: 253–262. sequence data: support for the recognition of Avenella and Vahlodea. JONSELL, B. 1978. Nomenclatural and taxonomic notes on Trisetum Pers. Taxon 56: 55–64. and Lophochloa Reichenb. (Gramineae). Botanical Journal of the CHOO, M. K., R. J. SORENG, AND J. I. DAVIS. 1994. Phylogenetic Linnean Society 76: 320–322. relationships among Puccinellia and allied genera of Poaceae as KELLOGG, E. A., R. APPELS, AND R. J. MASON-GAMER. 1996. When gene inferred from chloroplast DNA restriction site variation. American trees tell different stories, incongruent gene trees for diploid genera of Journal of Botany 81: 119–126. the Triticeae (Gramineae). Systematic Botany 21: 321–347. CLAYTON, W. D. 1975. Chorology of the genera of Gramineae. Kew KERGUE´ LEN, M. 1975. Les Gramineae (Poaceae) de la flore francaise. Essai Bulletin 30: 111–132. de mise au point taxonomique et nomenclatural. Lejeunia 75: 1–343. CLAYTON, W. D. 1981. Evolution and distribution of grasses. Annals of the KOCH, W. D. J. 1854. Synopsis florae germanicae et helveticae, vol. 2. Missouri Botanical Garden 68: 5–14. Friederici Wilmans, Francofurti, Germany. 964 pp. CLAYTON, W. D., AND S. A. RENVOIZE. 1986. Genera graminum, grasses of KOTSERUBA, V., D. GERNAND,A.MEISTER, AND A. HOUBEN. 2003. the world. Her Majesty’s Stationery Office, London, UK. 389 pp. Uniparental loss of ribosomal DNA in the allotetraploid grass DAVIS, J. I., AND R. J. SORENG. 1993. Phylogenetic structure in the grass Zingeria trichopoda (2n¼8). Genome 46: 156–163. family (Poaceae) as inferred from chloroplast DNA restriction site LEACHE´ , A. D., AND T. N. REEDER. 2002. Molecular systematics of the variation. American Journal of Botany 70: 1444–1454. eastern fence lizard (Sceloporus undulatus): a comparison of DEBRY, R. W., AND R. G. OLMSTEAD. 2000. A simulation study of reduced parsimony likelihood, and Bayesian approaches. Systematic Biology tree-search effort in bootstrap resampling analysis. Systematic Biology 51: 44–68. 49: 171–179. LEDEBOUR,K.F.VON. 1853. Flora rossica, vol. 4. Stuttgart, Germany. 741 EIG, A. 1929. Zwei neue Gramineengattungen von der Ventenata-Gattung pp. gesondert. Repertorium specierum novarum regni vegetabilis 26: 65– MACFARLANE, T. D. 1987. Poaceae subfamily Pooideae. In R. Sorderstrom, 79. K. W. Hilu, C. S. Campbell, and M. E. Barkworth [eds.], Grass FELSENSTEIN, J. 1985. Confidence limits on phylogenies: an approach using systematics and evolution. Smithsonian Institution, Washington, the bootstrap. Evolution 39: 783–791. D.C., USA. 473 pp. FINOT, V. L., P. M. PETERSON,R.J.SORENG, AND F. O. ZULOAGA. 2004. A MACFARLANE, T. D., AND L. WATSON. 1980. The circumscription of revision of Trisetum, Peyritschia,andSphenopholis (Poaceae: Poaceae subfamily Pooideae, with notes on some controversial Pooideae: Aveninae) in Mexico and Central America. Annals of the genera. Taxon 29: 645–666. Missouri Botanical Garden 91: 1–30. MACFARLANE, T. D., AND L. WATSON. 1982. The classification of Poaceae FINOT, V. L., P. M. PETERSON,R.J.SORENG, AND F. O. ZULOAGA. 2005a. A subfamily Pooideae. Taxon 31: 178–203. revision of Trisetum and Graphephorum (Poaceae: Pooideae: MAIRE, R., M. GUINOCHET, AND L. FAUREL. 1953. Flore de l’Afrique du Aveninae) in North of Me´xico. Sida 21(3): 1419– Nord, vol. 2. P. Lechevalier, Paris, . 374 pp. 1453. MASON-GAMER, R. J., AND E. A. KELLOGG. 1996. Testing for phylogenetic FINOT, V. L., P. M. PETERSON,F.O.ZULOAGA,R.J.SORENG, AND O. conflict among molecular data sets in the tribe Triticeae (Gramineae). MATTHEI. 2005b. A revision of Trisetum (Poaceae: Pooideae: Systematic Biology 45: 524–545. Aveninae) in South America. Annals of the Missouri Botanical MASON-GAMER,R.J.,N.L.ORME, AND C. M. ANDERSON. 2002. Garden 92: 533–568. Phylogenetic analysis of North American Elymus and the mono- FREY, L. 1999. Avenella: a genus of the Aveneae (Poaceae) worthy of genomic Triticeae (Poaceae) using three chloroplast DNA data sets. recognition. Fragmenta floristica et geobotanica. Supplement. 7: 27– Genome 45: 991–1002. 32. MOSULISHVILI, M. 2000. Directions of evolution of subtribe Koeleriinae GAUT, B. S., L. P. TREDWAY,C.KUBIK,R.L.GAUT, AND W. MEYER. 2000. (Gramineae). Bulletin of the Academy of Science 161(3): Phylogenetic relationships and genetic diversity among members of 496–498. September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1567

NADOT, S., R. BAJON, AND B. LEJEUNE. 1994. The chloroplast gene rps4 as a World grasses (Poaceae). IV. Subfamily Pooideae. Contributions tool for the study of Poaceae phylogeny. Plant Systematics and from the United States National Herbarium 48: 1–725. Evolution 191: 27–38. STACE, C. A. 2005. Plant and biosystematics—does DNA POSADA, D., AND K. A. CRANDALL. 1998. Modeltest: testing the model of provide all the answers? Taxon 54: 999–1007. DNA substitution. Bioinformatics 14: 817–818. STEBBINS, G. L. 1956. Cytogenetics and evolution of the grass family. PRAT, H. 1960. Vers une classification naturelle des gramine´es. Bulletin de American Journal of Botany 43: 890–905. la Socie´te´ Botanique de France 107: 32–79. STEBBINS, G. L., AND B. CRAMPTON. 1961. A suggested revision of the grass QUINTANAR, A., P. CATALA´ N, AND S. CASTROVIEJO. 2006. Adscription of genera of temperate North America. Recent Advances in Botany 1: Parafestuca albida (Lowe) E.B. Alexeev to Koeleria Pers. Taxon 55: 133–145. 664–670. SUBBOTIN, S. A., E. L. KRALL,I.T.RILEY,V.N.CHIZHOV,A.STAELENS,M. RAMBAUD, A. 1996. Sequence lignment editor (Se-Al), version 1.0 alpha 1. DE LOOSE, AND M. MOENS. 2004. Evolution of the gall-forming plant [Computer program] Oxford University, Oxford, UK. parasitic nematodes (Tylenchida: Anguinidae) and their relationships REEDER, J. R. 1953. Affinities of the grass genus Beckmannia Host. with hosts as inferred from internal transcribed spacer sequences of Bulletin of the Torrey Botanical Club 80: 187–196. nuclear ribosomal DNA. Molecular Phylogenetics and Evolution 30: REICHENBACH, H. G. L. 1830. Flora germanica excursoria, vol. 1. Leipzig, 226–235. Germany. 434 pp. SWOFFORD, D. L. 2002. PAUP*: phylogenetic analysis using parsimony RODIONOV, A. V., N. B. TYUPAL,E.C.KIM,E.M.MACHS, AND I. G. (*and other methods), version 4.0 beta 10. [Computer program]. Sinauer, Sunderland, Massachusetts, USA. LOKUSTOV. 2005. Genome composition of the autotetraploid oat species Avena macrostachya determined by comparative analysis of TABERLET, P., L. GIELLY,G.PATOU, AND J. BOUVET. 1991. Universal primers the ITS1 and ITS2 sequences: on the oat karyotype evolution on early for amplification of three non-coding regions of chloroplast DNA. events of oats species divergence. Russian Journal of Genetics 41(1): Plant Molecular Biology 17: 1105–1109. 1–11. TEMPLETON, A. R. 1983. Phylogenetic inference from restriction endonu- clease cleavage site maps with particular reference to the evolution of ROMERO GARCI´A, A. T., G. BLANCA LO´ PEZ, AND C. MORALES TORRES. 1988. humans and the apes. Evolution 37: 221–244. Revisio´n del ge´nero Agrostis L. (Poaceae) en la Penı´nsula Ibe´rica. THOMPSON, J. D., T. J. GIBSON,F.PLEWNIAK,F.JEANMOUGIN, AND D. G. Ruizia 7: 1–160. HIGGINS. 1997. The ClustalX windows interface: flexible strategies for ROMERO ZARCO, C. 1984. Revisio´n taxono´mica del ge´nero Avenula multiple sequence alignment aided by quality analysis tools. Nucleic (Dumort.) Dumort. (Gramineae) en la Penı´nsula Ibe´rica e Islas Acids Research 24: 4876–4882. Baleares. Lagascalia 13: 39–146. TORRECILLA, P., AND P. CATALA´ N. 2002. Phylogeny of broad-leaved and ROMERO ZARCO, C. 1996. Contribucio´n al conocimiento de las gramı´neas fine-leaved Festuca lineages (Poaceae) based on nuclear ITS del N de Marruecos. Lagascalia 18: 310–321. sequences. Systematic Botany 27: 241–251. RO¨ SER, M. 1989. Karyologische, systematische und chorologische TORRECILLA,P.,J.A.LO´ PEZ RODRI´GUEZ, AND P. CATALA´ N. 2004. Untersuchungen an der Gattung Helictotrichon Besser ex Schultes Phylogenetic relationships of Vulpia and related genera (Poeae, & Schultes im westlichen Mittelmeergebiet. Dissertationes Botanicae Poaceae) based on analysis of ITS and trnL-F sequences. Annals of 145: 1–250. the Missouri Botanical Garden 91: 124–158. ¨ ROSER, M. 1997. Patterns of diversification in the Mediterranean oat TORRECILLA, P., J. A. LO´ PEZ RODRI´GUEZ,D.STANCIK, AND P. CATALA´ N. 2003. grasses (Poaceae: Aveneae). Lagascalia 19: 101–120. Systematics of Festuca sects. Eskia Willk., Pseudatropis Kriv., SCRIBNER, F. L. 1906. The genus Sphenopholis. Rhodora 8: 137–146. Amphigenes (Janka) Tzvel., Pseudoscariosa Kriv., and Scariosae SOLTIS, D. E., AND R. K. KUZOFF. 1995. Discordance between nuclear and Hack. based on analysis of morphological characters and DNA chloroplast phylogenies in the Heuchera group (Saxifragaceae). sequences. Plant Systematics and Evolution 239: 113–139. Evolution 49: 727–742. TUTIN, T. G., V. H. HEYWOOD,N.A.BURGES,D.M.MOORE,D.H. SORENG, R. J., AND J. I. DAVIS. 2000. Phylogenetic structure in Poaceae VALENTINE,S.M.WALTERS, AND D. A. WEBB [EDS.], 1980. Flora subfamily Pooideae as inferred from molecular and morphological Europaea, vol. 5, Poaceae. Cambridge University Press, Cambridge, characters: misclassification versus reticulation. In S. W. L. Jacobs UK. 452 pp. and J. Everett [eds.], Grasses: systematics and evolution. CSIRO TZVELEV, N. N. 1976. Zlaki SSSR. Nauka Publishers, Leningrad, . Publishing, Collingwood, Victoria, Australia. 416 pp. [English translation: 1983. Grasses of the Soviet Union. Amerid SORENG, R. J., J. I. DAVIS, AND J. J. DOYLE. 1990. A phylogenetic analysis Publishing, New Delhi, India. 1196 pp.] of chloroplast DNA restriction site variation in Poaceae subfam. TZVELEV, N. N. 1989. The system of grasses and their evolution. Botanical Pooideae. Plant Systematics and Evolution 172: 83–97. Review 55: 141–204. SORENG, R. J., P. M. PETERSON,G.DAVIDSE,E.J.JUDZIEWICZ,F.O. WATSON, L., AND M. J. DALLWITZ. 1992. The grass genera of the world. ZULOAGA,T.S.FILGUEIRAS, AND O. MORRONE. 2003. Catalogue of New CAB International Wallingford, Oxon, UK. 1081 pp.

APPENDIX. Voucher information and GenBank accession numbers for taxa used in this study. A dash indicates that the region was not sampled. Voucher specimens belong to the following collections: AQ ¼ Alejandro Quintanar collection; ARAN ¼ Herbario de la Sociedad de Ciencias Aranzadi; CA ¼ Carlos Aedo collection; CN ¼ Carmen Navarro collection; ES ¼ Elvira Sauquillo collection; JACA ¼ Herbario del Instituto Pirenaico de Ecologı´a de Jaca; MA ¼ Herbario del Real Jardı´n Bota´nico de Madrid; MERC ¼ Herbario de la Universidad de Me´rida (Venezuela); MP ¼ Manuel Pimentel collection; MS ¼ Miguel Sequeira collection; PC ¼ Pilar Catala´n collection; RS ¼ Robert Soreng collection; SC ¼ Santiago Castroviejo collection; UZ ¼ Herbario de la Universidad de Zaragoza.

Taxon—GenBank accessions: ITS, trnL-F, trnT-L; Voucher specimen or data source. Agrostis castellana Boiss. & Reut.—DQ539591, —, —; Spain: (1988), JACA 348895. Airopsis tenella (Cav.) Coss. & Durand— Guadalajara (1997), MA 648799. A. magellanica Lam.—AY705883, DQ539582, DQ631445, DQ631511; Portugal: Mogadouro (1997), —, —; Gardner et al. (unpublished). A. stolonifera L.—DQ336815, JACA 62597. Alopecurus geniculatus L.—DQ539571, DQ631433, DQ336835, DQ336860; Spain: Huesca: Ordesa (1996), JACA 380196. DQ631499; Spain: Guadalajara, El Cubillo de Uceda (1997), MA A. truncatula Parl. subsp. commista Castrov. & Charpin—DQ539592, 642779. A. vaginatus (Willd.) Pall. ex Kunth—Z96920 & Z96921, —, —, —; Spain: A Corun˜a, Montes do Pindo (1994), MA 581339. Aira —;Grebensteinetal.(1998);Caucasus,Ossethi.Ammochloa cupaniana Guss.—DQ631442, DQ631508; Spain: Jae´n, La Carolina palaestina Boiss.—DQ539587, DQ631451, DQ631517; Spain: 1568 AMERICAN JOURNAL OF BOTANY [Vol. 94

Almerı´a, Tabernas (1986), MA 478222. Ammophila arenaria (L.) Buschm.—DQ539616, DQ631480, DQ631547; Portugal: Madeira: ca. Link—DQ539590, DQ631456, DQ631522; Spain: A Corun˜a, Carballo Pico Arieiro (2004), MS 4507. D. setacea (Huds.) Hack.—DQ539615, (2004), MP s/n. Anthochloa lepidula Nees & Meyen—DQ539566, DQ631479, DQ631546; Spain: Lugo, Vilalba (2000), MA 653850. D. DQ631430, DQ631496; Bolivia: Dpto. La Paz, Cumbre near La Paz tenella Petrie—AY752475, —, —; Gardner et al. (unpublished). (2002), MA 721313. Anthoxanthum amarum Brot.—DQ539584, Deyeuxia lacustris Edgar & Connor—AY705887, —, —; Gardner et DQ631448, DQ631514; Spain: Asturias, La Coba (1994), MA 539163. al. (unpublished). Dielsiochloa floribunda (Pilg.) Pilg.—DQ539563, A. aristatum Boiss.—DQ539585, DQ631449, DQ631515; Spain: DQ631428, DQ631494; Bolivia: Dpto. La Paz, Cumbre near La Paz Pontevedra, Cabo Silleiro, ES s/n. Antinoria agrostidea (DC.) Parl.— (2002), MA 721312. Dissanthelium calycinum (J. Presl) Hitchc.— DQ539562, —, —; Spain: Zamora, Ta´bara (1996), MA 651156. Apera DQ539567, DQ631431, DQ631497; Bolivia: Dpto. La Paz, Cumbre interrupta (L.) P. Beauv.—DQ539570, —, —; Spain: Huesca (1988), near La Paz (2002), MA 721311. JACA 167088. Arrhenatherum elatius (L.) P. Beauv. ex J. & C. Presl Echinaria capitata (L.) Desf.— —, DQ631453, DQ631519; Spain: subsp. bulbosum (Willd.) Schu¨bl. & Martens—DQ336821, Murcia, Moratalla (1997), MA 591692. DQ336841, DQ336866; Spain: Zaragoza (1999), JACA 128099. A. Festuca arundinacea (P. Beauv.) Schreber—AF519976, AY098995, calderae A. Hansen—DQ539596, DQ631462, DQ631528; Spain: DQ367405; Spain: Lugo, La´ncara, Catala´n et al. (2004). F. frigida Canarias, Tenerife (2003), SC 17370. Avellinia michelii (Savi) Parl.— (Hack.) K. Richt.—AF478481, AF478521, DQ631485; Spain: —, DQ631465, DQ631531; Spain: Huesca, Monzo´n (1991), JACA Granada, Veleta, Catala´n et al. (2004). F. borderei (Hack.) K. 304591. Avena barbata Pott ex Link—AY093613, —, —; Moore and Richt.—AF303403, AF478510, DQ631484; Spain: Huesca, Field (2005). A. clauda Durieu—AY522432, —, —; Rodionov et al. Vallibierna, Torrecilla & Catala´n (2002). F. fontqueri St.-Yves— (2005), Azerbaijan. A. eriantha Durieu—DQ336822, DQ336842, AF303404, AF533044, DQ631486; : Rif Mountains, DQ336867; Spain: Madrid, Chincho´n (2001); UZ JARL 032001. A. Torrecilla & Catala´n (2002). F. ki ngii (S. Watson) Cassidy— hirtula Lag.—AY522435, —, —; Rodionov et al. (2005), Israel. A. AF303410, AY099004, DQ631487; USA: , Boulder Co, longiglumis Durieu—DQ539597, DQ631463, DQ631529; France: Flat Irons, Catala´n et al. (2004). F. ovina L.—AF532959, AF533063, Nice (1986), JACA 428986. A. macrostachya Balansa ex Coss. & DQ367406; Germany: Thu¨ringen, Saale-Holzland-Kreis, Catala´n et al. Durieu—AY522433, —, —; Rodionov et al. (2005), . A. pilosa (2004). F. paniculata (L.) Schinz. & Thell. subsp. paniculata— Scop.—AY530162, —, —; Rodionov et al. (2005), Azerbaijan. A. AF303407, AF533046, DQ336858; France: Mont Aigoual, Torrecilla sativa L.—AY520821, —, —; Rodionov et al. (2005), Germany. A. &Catala´n (2002). F. rivularis Boiss.—AF478475, AF478512, ventricosa Balansa ex Coss.—AY522437, —, —; Rodionov et al. DQ631488; Spain: Huesca, Cotiella, Torrecilla & Catala´n (2002). F. (2005), Cyprus. Avenula albinervis (Boiss.) M. Laı´nz—AJ389123 & rubra L.—AY118088, AY118099, DQ336857; : Valais, AJ389124, —, —; Hemleben et al. (unpublished). A. bromoides Desses des Ferret, Catala´n et al. (2004). F. triflora Desf.—AF538362, (Gouan) H. Scholz— —, DQ631459, DQ631525; Spain: Huesca, AF533052, DQ631483; Spain: Granada, Grazalema, Catala´n et al. Valfarta (1995), JACA 75295. A. bromoides—Z96844 & Z96845, —, (2004). —; Grebenstein et al. (1998); France, Maury. A. compressa (Heuff.) Gastridium ventricosum (Gouan) Schinz & Thell.—DQ336817, W. Sauer & H. Chmelitschek—Z96848 & Z96849, —, —; DQ336837, DQ336862; Spain: Baleares, Mallorca, Puigpunyent Grebenstein et al. (1998); Turkey, Vil. Bolu. A. sulcata (Gay ex (1998), MA 618134. Gaudinia fragilis (L.) P. Beauv. (1)— Boissier) Dumort.—DQ539595, DQ631461, DQ631527; Spain: DQ539600, DQ631467, DQ631533; Spain: Ourense, O Barco de Ciudad Real, Fuencaliente (1996), MA 597123. A. pratensis (L.) Valdeorras (1989), MA 517046. G. fragilis (2)— —, DQ631478, Dumort.—Z96860 & Z96859, —, —; Grebenstein et al. (1998); DQ631545; Spain: Ciudad Real, Fuencaliente (1996), MA 597178. Caucasus, Pikritis Khevsureti. A. pubescens (Huds.) Dumort.— —, Graphephorum wolfii (Vasey) Vasey ex Coult.—DQ336823, DQ631460, DQ631526; Spain: Huesca, Cerler (1997), JACA 177197. DQ336843, DQ336868; USA: California, Sierra Nevada (2004), RS A. pubescens—Z96876 & Z96877, —, —; Grebenstein et al. (1998); 7416. Gymnachne koelerioides (Trin.) Parodi—DQ539594, Caucasus, Ossethi. DQ631458, DQ631524; (2001), RS 7035. (L.) Host—AJ389163, —, —; Hemleben et al. Helictotrichon convolutum (C. Presl) Henrard—Z96820 & Z96821, —, (unpublished). Brachypodium distachyon (L.) Beauv.—AF303399, —; Grebenstein et al. (1998); Dalmatia. H. desertorum (Less.) Pilg.— AF478500, DQ336855; : Ljubljana, Torrecilla & Catala´n AJ389095 & AJ389096, —, —; Hemleben et al. (unpublished). H. (2002). Briza media L.—DQ539583, DQ631446, DQ631512; Spain: filifolium (Lag.) Henrard—DQ336819, DQ336839, DQ336864; Huesca: Panticosa (2000) PC s/n. B. minor L.—L36510, —, —; Hsiao Spain: Almerı´a (1997), MA 591453. H. jahandiezii (Litard. ex et al. (1995). Bromus tectorum L.—AJ608154, —, —; Blattner (2004). Jahand. & Maire) Potztal—Z96840 & Z96841, —, —; Grebenstein Calamagrostis arundinacea (L.) Roth— —, DQ631455, DQ631521; et al. (1998); Morocco, Moyen Atlas. H. sedenense (Clar. ex Lam & Spain: Navarra, Orbaitzeta (2001), ARAN 64564. C. epigejos (L.) DC.) Holub—DQ336820, DQ336840, DQ336865; Spain: Huesca Roth—AJ306449, —, —; Jakob & Blattner (unpublished). (1997), JACA 177297. Hellerochloa fragilis (Luces) Rauschert— Chaetopogon fasciculatus (Link) Hayek—DQ539593, DQ631457, AF532960, AF533059, DQ631492; Venezuela: Me´rida, Pa´ramo de DQ631523; Spain: Ca´ceres, Torrejo´n el Rubio (1983), MA 252874. Piedras Blancas, MERC, PC s/n. Hierochloe australis (Schrad.) (L.) P. Beauv.—DQ539565, DQ631429, Roem. & Schult.— —, DQ631447, DQ631513; Finland: South Ha¨me DQ631495; Spain: Huesca, Sallent, Portalet (2004), UZ, PC s/n. (1991), MA 696177. H. equiseta Zotov—AY705901, —, —; Gardner Cinna latifolia (Trevir. ex Go¨pp.) Griseb.—DQ539569, DQ631432, et al. (unpublished). H. fusca Zotov—AY705902, —, —; Gardner et DQ631498; Finland: South Ha¨me (1982), MA 363675. Colpodium al. (unpublished). H. novae-zelandiae Gand.—AY705900, —, —; versicolor (Steven) Schmalh.—AY497472, —, —; Russia: Teberda, Gardner et al. (unpublished). Holcus gayanus Boiss.—DQ539574, Rodionov et al. (2005). Corynephorus canescens (L.) P. Beauv.— DQ631436, DQ631502; Spain: Asturias, Puente del Infierno (1998), DQ539578, DQ631440, DQ631506; Spain: Soria, Min˜o de Medinaceli MA 655816. H. lanatus L.—DQ539575, DQ631437, DQ631503; (2004), AQ 1079. echinatus L.—AF532937, AF533031, : Abruzzo, Sorgente del Tirino (2002), MA 699215. DQ631482; Spain: Soria, Monte Valonsadero, Catala´n et al. (2004). Koeleria albescens DC.—DQ336824, DQ336844, DQ336870; Spain: A glomerata L.—AF393013, AF533028, DQ631481; Spain: Corun˜a (2003), MA 706574. K. crassipes Lange (1)—DQ539603, Zaragoza, Moncayo, Torrecilla & Catala´n (2002). Deschampsia DQ631469, DQ631535; Spain: Madrid (2003), MA 706575. K. antartica E. Desv.—AF521900, —, —; Corach et al. (unpublished). crassipes (2)—DQ539602, —, —; Spain: Madrid (2003), MA D. cespitosa (L.) P. Beauv.—DQ539579, DQ631441, DQ631507; 706580. K. dasyphylla Willk.—DQ336825, DQ336845, DQ336871; , Pont de Capigol (2002), MA 700212. D. chapmanii Petrie— Spain: Ca´diz (1993), MA 526298. K. macrantha (Ledeb.) Schult.— AY752476, —, —; Gardner et al. (unpublished). D. flexuosa (L.) DQ336826, DQ336846, DQ336872; Spain: Huesca (2001), JACA Trin.—DQ539577, DQ631439, DQ631505; Andorra, Puerto de 264664. K. pyramidata (Lam.) P. Beauv.—DQ336827, DQ336847, Envalira (2002), MA 689503. D. maderensis (Hackel & Bornm.) DQ336873; Spain: Lleida (1999), JACA 147297. K. splendens C. September 2007] QUINTANAR ET AL.—PHYLOGENY OF TRIBE AVENEAE 1569

Presl—DQ336828, DQ336848, DQ336874; Italy (2000), MA 645409. DQ336854, DQ336880; Spain: Almerı´a (1998), MA 613459. R. K. vallesiana (Honck.) Gaudin subsp. vallesiana (1)—DQ336829, salzmannii (Boiss. & Reut.) Holub—DQ539613, DQ631476, DQ336849, DQ336875; Spain: Madrid (2003), MA 706578. K. DQ631542; (1999), MA 693894. vallesiana subsp. vallesiana (2)—DQ539604, DQ631470, Secale cereale L.—AF303400, AF478501, DQ336856; USA: Torrecilla DQ631536; Spain: Palencia (1995), MA 560050. K. vallesiana & Catala´n (2002). Sesleria argentea (Savi) Savi—AF532931, subsp. castellana (Boiss. & Reut.) Domin—DQ539601, DQ631468, AF533030, DQ631544; Spain: Navarra, Araxes, Catala´n et al. DQ631534; Spain: Madrid, Aranjuez (2004), AQ 997. (2004). S. coerulea (L.) Scop.—DQ539586, DQ631450, Lagurus ovatus L.—DQ539598, DQ631464, DQ631530; Spain: Almerı´a, DQ6315106; Spain: Huesca, Pue´rtolas (2001), JACA 266634. Cuevas del Almanzora (1998), MA 613465. Lamarckia aurea (L.) Sclerochloa. dura (L.) P. Beauv.—AF532933, AF533023, —; Moench—AF532935, AF533029, DQ631490; Spain: Zaragoza, Spain: Segovia, Sepu´lveda, Catala´n et al. (2004). Scolochloa Puente Almozara (2000), Catala´n et al. (2004). Lolium perenne festucacea (Willd.) Link—DQ539564, —, —; Finland: Joutsa L.—AF303401, AF478504, DQ367404; England (cv.), Torrecilla & (1992), MA 692842. (Rydb.) Rydb.— Catala´n (2002). DQ539599, DQ631466, DQ631532; USA: Kentucky, Dobertson Co. Mibora minima (L.) Desv.—DQ539589, DQ631454, DQ631520; Spain: (1995), MA 721314. Sphenopus divaricatus (Gouan) Reichenb.— Madrid, Boadilla del Monte (2004), AQ 977. Milium effusum L.— AF532939, AF533033, DQ631493; Spain: Zaragoza, Vedado de DQ539573, DQ631435, DQ631501; Finland: Lapponia sompiensis, Pen˜aflor, Catala´n et al. (2004). Sodankyla¨ (1996), JACA 199998. Triplachne nitens (Guss.) Link—DQ336816, DQ336836, DQ336861; Oreochloa disticha (Wulfen) Link—DQ539588, DQ631452, DQ631518; Spain: Almerı´a, Playa de Carboneras (1982), MA 292711. Trisetum Spain: Palencia, Cervera de Pisuerga (1997), MA 590914. baregense Laffitte & Mie´geville—DQ539605, DQ631471, Parafestuca albida (Lowe) E.B. Alexeev—AF532930, AF533022, DQ631537; Spain: Huesca (1998), JACA 120998. T. drucei Edgar— DQ336869; Portugal: Madeira, Pico do Arieiro (2001), MA 721307. AY752485, —, —; Gardner et al. (unpublished). T. dufourei Boiss.— Parapholis incurva (L.) C.E. Hubb.—AF532942, AF533036, DQ539606, —, —; Spain: Ca´diz (1993), JACA 284999. T. flavescens DQ631491; Spain: Zaragoza, Vedado de Pen˜aflor, Catala´n et al. (L.) P. Beauv.—DQ336830, DQ336850, DQ336877; (2004), (2004). Periballia involucrata (Cav.) Janka—DQ539576, DQ631438, CA 10141. T. glaciale Boiss.—DQ539614, DQ631477, DQ631543; DQ631504; Spain: Ciudad Real, Solana del Pino (1996), MA 597226. Spain: Granada, Pico Veleta (1986), MA 398253. T. gracile (Moris) Phalaris canariensis L.—DQ539580, DQ631443, DQ631509; Spain: Boiss.—DQ539607, DQ631472, DQ631538; Italy: Sardegna (2003), Huesca, Cuarte (cultivated), AQ 1429. P. coerulescens Desf.— . T. hispidum Lange—DQ336831, DQ336851, DQ336376; DQ539581, DQ631444, DQ631510; Spain: Ca´ceres, Malpartida de SC 17158 Plasencia (2001), MP s/n. P. truncata Guss. ex Bertol.—L36522, —, Spain: Leo´n, Valverde de la Sierra (1994), MA 542627. T. —; Hsiao et al. (1995). Phleum phleoides (L.) H. Karst.—AF498396, loeflingianum (L.) P. Beauv.—DQ539608, DQ631473, DQ631539; —, —; Subbotin et al. (2004). P. pratense L. subsp. bertolonii (DC.) Spain: Huesca (1996), JACA 307296. T. ovatum Pers.—DQ336832, Bornm.—DQ539568, —, —; Spain: Ciudad Real, Fuencaliente DQ336852, DQ336878; Spain: Toledo, Hinojosa de San Vicente (1996), MA 597217. L.—AF521901, —, —; Corach et (1995), MA 556705. T. paniceum (Lam.) Porsild—DQ539609, al. (unpublished). P. infirma Kunth—AF393012, AF488773, DQ631474, DQ631540; Spain: Jae´n, Andu´jar (2003), MA 652453. T. DQ367407; Spain: Zaragoza, La Jota, Torrecilla & Catala´n (2002). spicatum (L.) K. Richt.—AY752486,—,—; Gardner et al. Polypogon maritimus Willd.—DQ336818, DQ336838, DQ336863; (unpublished). T. tenellum (Petrie) A.W. Hill—AY752487, —, —; Spain: Ciudad Real, Valverde (1999), MA 648807. Gardner et al. (unpublished). T. turcicum Chrtek—Z96902 & Z96903, Pseudarrhenatherum longifolium (Thore) Rouy—AJ389161 & —, —; Grebenstein et al. (1998); Caucasus, Dzhavachethi. T. youngii AJ389162, —, —; Hemleben et al. (unpublished). Puccinellia Hook. f.—AY752488, —, —; Gardner et al. (unpublished). distans (L.) Parl.—AF532934, AF533024, DQ336859; Spain: Ventenata dubia (Leers) Cosson—DQ539572, DQ631434, DQ631500; Navarra, Lazagurrı´a, Catala´n et al. (2004). Bulgaria (2004), CN 5025. Vulpia myuros (L.) C.C. Gmel.— Rostraria cristata (L.) Tzvelev—DQ336833, DQ336853, DQ336879; AY118092, AY118103, DQ631489; USA: Washington, Seattle, Spain: Tarragona (1999), JACA 630099. R. hispida (Savi) Dogan— Lake Forest Park, Catala´n et al. (2004). DQ539610, —, —; Spain: Sevilla (1977), MA 278005. R. litorea (All.) Wangenheimia lima (L.) Trin.—AF478498, AF478536, —; Spain: Holub—DQ539611, —, —; France: Corse (1981), MA 392462. R. Zaragoza, Vedado de Pen˜aflor, Catala´n et al. (2004). obtusiflora (Boiss.) Holub—DQ539612, DQ631475, DQ631541; Zingeria trichopoda (Boiss.) P. A. Smirn.—AJ428835, —, —; Kotseruba Israel (1989), MA 498402. R. pumila (Desf.) Tzvelev—DQ336834, et al. (2003).